Bovine immunodeficiency virus (BIV) based vectors

ABSTRACT

This invention pertains to BIV constructs encompassing BIV combination vectors, BIV vectors and BIV packaging vectors and particularly the invention pertains to a three vector system comprising: a) a BIV vector construct including a DNA segment from a BIV genome, a packaging sequence to package RNA into virions; a promoter operably linked to the DNA segment; and a transgene operably linked to a second promoter; b) a BIV packaging vector construct comprising a BIV DNA sequence fragment comprising at least a gag gene or pol gene of BIV; a promoter operably linked to the BIV DNA fragment; and a polyadenylation sequence located downstream of the BIV DNA fragment; and c) an expression vector construct comprising a gene encoding a viral surface protein. Also provided is a method for transferring a gene of interest into a mammalian cell.

This application claims the benefit under 35 USC §119(e) of thefollowing U.S. provisional patent applications: (1) ProvisionalApplication No. to be assigned, filed Dec. 14, 1999, as application Ser.No. 09/464,460 for “Bovine Immunodeficiency Virus (BIV) Based Vectors,”and subject to a Petition for Conversion to Provisional Application,filed Nov. 16, 2000; and (2) Provisional Application No. 60/249,492,filed Nov. 17, 2000 for “Bovine Immunodeficiency Virus (BIV) BasedVectors.” The disclosures of these two provisional applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to use of recombinant viruses asvectors, and more specifically to recombinant bovine immunodeficiencyvirus (BIV) based vector constructs capable of expressing a desiredprotein in target cells.

The use of recombinant virus vectors in a variety of applicationsincluding, for example, gene therapy requires that the virus not becapable of replication in the target cells to avoid the possibility ofuncontrolled virus or cell proliferation. In addition to safety, therecombinant virus vector system must be efficient and accurate.

Retroviruses have been used as vectors to mediate gene transfer intoeukaryotic cells. Retroviruses are RNA viruses that include thesubfamilies lentivirus, spumavirus, and oncovirus. These viruses canreplicate and integrate into a host cell genome through a DNAintermediate, generally called a provirus.

The viral vectors are generally constructed such that the majority ofthe viral genes are deleted and replaced by a gene of interest. Mostfrequently the gene of interest is transcribed under the control of theviral regulatory sequences within the long terminal repeat (LTR).Alternatively, the gene of interest may be expressed under theregulation of its own internal promoter. The genes which have beendeleted from the vector are generally provided by one or more helper orpackaging constructs in a packaging cell line (Bender et al., J. Virol.61: 1639-1649 (1987) and Miller et al., Biotechniques, 7:980-990(1989)). Also see Markowitz et al., J. Virol. 62:1120-1124 (1988)wherein complementary portions of the helper construct were divided ontwo separate constructs. The packaging cell line may be transfected withthe retroviral vector, thereby producing vector RNA that is packagedinto the virus particles. These released virus particles are replicationdefective and can be used to deliver the retroviral vector carrying aheterologous gene of interest to target cells.

To increase safety, efficiency and accuracy of the recombinant vectorsystems, various improved recombinant systems have been constructed. Onetype of improvement includes making safer packaging cell lines that aregenerated by deletions in the 3′ Long Terminal Repeat (LTR). Otherimprovements include increasing the host range by replacement of oneviral env gene with that of another viral env gene, thereby creating ahybrid producer line that generates pseudotyped helper viruses. Morespecifically, HIV has been given an extended host cell range bypseudotyping with the unrelated viruses VSV and HSV (Zhu et al., J.AIDS, 3:215-219 (1990) and Naldini et al., Science, 272:263-267,(1996)). Further improvements have been made by the use of minimum viralcoding regions in the vector. Additionally, most packaging cell linescurrently in use have been transfected with separate plasmids, eachcontaining one of the necessary coding sequences so that multiplerecombination events are necessary before replication competent viruscan be produced.

In contrast to vectors derived from oncoviruses, lentivirus can infectnondividing cells. This property is especially useful for in vivo genetherapy.

BIV generally does not infect human cells and, therefore, the use of aBIV genomic backbone in the vectors of the present invention overcomesdifficulties of prior packaging cell lines and further provides otherrelated advantages for improved vector construction.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a BIV combination vectorconstruct comprising a DNA segment from a BIV genome, wherein the DNAsegment comprises a gag gene, a pol gene, a segment of the env gene, anda BIV packaging sequence to package BIV RNA into virions; a promoteroperably linked to the DNA segment; and a transgene operably linked tothe promoter wherein the transgene is inserted into the segment of theenv gene. In a preferred embodiment, a part of the interior env gene isdeleted, and the transgene is inserted into the gap created by thedeletion. In further embodiments, the DNA segment may include one ormore of the following genes vif, vpw, vpv, rev, and tat, particularlytat and rev.

The invention also provides a BIV vector comprising a DNA segment from aBIV genome, a packaging sequence to package RNA into virions; a firstpromoter operably linked to the DNA segment; and a transgene operablylinked to a second promoter. In one preferred embodiment, the packagingsequence is a BIV packaging sequence and the promoter is a LTR promoteror a cytomegalovirus (CMV) promoter. In a further preferred embodiment,the DNA segment also comprises a portion of a gag gene. In anotherembodiment, the vector includes a rev-response element (RRE).

Further provided by this invention is a BIV packaging constructcomprising a BIV DNA sequence fragment including at least a gag or polgene of BIV; and a promoter operably linked to the BIV DNA fragment.Preferably, a polyadenylation sequence is located downstream of the BIVDNA fragment. In a further embodiment, the packaging construct includesan internal ribosome binding site (IRES) and preferably includes aheterologous intron.

The invention also provides a three vector system comprising: (1) a BIVvector construct including a DNA segment from a BIV genome, a packagingsequence to package RNA into virions; a promoter operably linked to theDNA segment; and a transgene operably linked to a second promoter; (2) aBIV packaging vector construct including a BIV DNA sequence fragmentincluding at least a gag or pol gene of BIV; a promoter operably linkedto the BIV DNA fragment; and a polyadenylation sequence locateddownstream of the BIV DNA fragment; and (3) an expression vectorincluding a gene encoding a viral surface protein. In one embodiment,the viral surface protein is a vesicular stomatitis virus (VSV)-Genvelope glycoprotein. In a second embodiment, the DNA segment of theBIV vector construct includes a portion of a BIV gag gene. In furtherembodiments, the BIV vector construct includes one or more BIV genesselected from the group consisting of gag, vif, vpw, vpv, tat, rev, andenv.

Further provided by this invention is a method of transferring a gene ofinterest to a mammalian cell obtained by transfecting a eukaryotic hostcell with the three vector system as claimed and disclosed herein;culturing the transfected host cell; collecting the virions produced;and administering the virions to a mammalian cell to allow infection ofthe mammalian cell and transferring the gene of interest. In oneembodiment, the mammalian cell is located in vitro, and in anotherembodiment the mammalian cell is located in vivo.

The invention further provides for a two vector system, including afirst vector construct comprising, a DNA segment from a BIV genome,wherein the DNA segment comprises gag and pol genes, a BIV packagingsequence to package BIV RNA into virions, a promoter operably linked tothe DNA segment, and a transgene operably linked. to the promoter; and aviral surface protein expression vector. Preferably, such vector is avesicular stomatitis virus (VSV)-G. envelope glycoprotein expressionvector.

Further provided by this invention is a method of transferring a gene ofinterest to a mammalian cell by transfecting a eukaryotic host cell withthe two vector system as claimed and disclosed herein; culturing thetransfected host cell and collecting the virions produced; andadministering the virions to a mammalian cell to allow infection of saidcell and transfer of the gene of interest.

A further, particularly preferred embodiment of this invention providesa minimal BIV-based vector system. The system comprises a minimal vectorconstruct, a minimal packaging. construct, and a minimal viral surfaceprotein expression construct. The minimal vector construct comprises apromoter linked to a first BIV R region, a BIV U5 element linked to thefirst BIV R region, a packaging sequence, a transgene, and a BIV U3element linked to a second BIV R region, wherein the promoter initiatestranscription of the vector. The packaging construct comprises apromoter operatively linked to a BIV gag/pol coding sequence and apolyadenylation signal at the 3′. end. of the gag/pol coding sequence.The viral surface protein expression construct comprises a promoteroperatively linked to a viral envelope coding sequence and apolyadenylation signal at the 3′ end of the coding sequence.

This embodiment of the invention further comprises a packaging cell. Thepackaging cell comprises the minimal packaging construct and the minimalviral surface protein expression construct.

The addition of the minimal vector construct results in a producer cell.The producer cell produces virions that contain the vector. Infecting acell with the virions results in the transfer of the transgene to thecell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic illustration of wild-type BIV proviral plasmid (pBIV)clone 127.

FIG. 2: BIV packaging constructs: Wild-type BIV, CMV-driven BIV, andthree packaging constructs (BH1-3) are depicted for their gag, pol, andenv genes; accessory genes are not shown. Also depicted are the viralmajor splice donor (MSD), a small chimeric intron (in), the HIV-1rev-response element (RRE), the puromycin N-acetyltransferase cDNAlinked to an internal ribosome entry site from encephalomyocarditisvirus (IRES-puro), and the SV40 late polyadenylation signal (SV40polyA).

FIG. 3: BIV vectors and transduction efficiencies. All vectors containthe CMV immediate-early promoter, ending in the TATA box, linked to theBIV 5′ LTR starting immediately after the TATA box. BIV sequencesterminate at the gag start codon (BCCG and BCPG) or approximately 510 bpinto the gag coding region (all others). All vectors contain the eGFPcDNA linked to a heterologous, “internal” promoter: CMV, PGK, or MND.Some vectors contain one or more insertions between the BIV 5′ segmentand the internal promoter; these insertions include a potential BIVcentral polypurine tract (cPPT), the 5′ segment of the gag gene, thebeta-interferon scaffold attachment region (SAR), and the putative BIVrev-response element (RRE). Downstream of the eGFP cDNA lies the BIV 3′LTR and approximately 130 bp (BCCG and BCPG), 1.2 kb (BC2CG, BC2PG, andBC2MG) or 80 bp (all others) of adjacent env sequences. Two vectors(BC4MG and BC4MGppt) contain modified 3′ LTRs in which most of the 3′LTR U3 region is replaced by the SV40 late polyadenylation signalenhancer element (“SINSV”). Transcription start sites and directions areindicated with arrows. At the bottom are depicted two HIV-1 controlvectors used in a series of experiments. At the right are thetransduction efficiencies of the vectors in 293T cells 3 dayspost-infection, using packaging construct BH2 and VSV-G, in a series ofexperiments. Infections from experiments numbers 3-5 are performed inthe presence of an additional buffer to retard pH elevation duringspinoculation. As a result, transduction efficiencies are elevated.

FIG. 4. Schematic illustration of the BIV based gene transfer systemconsists of packaging construct, vector backbone, and VSV-G expressionconstruct. CMV: CMV early promoter; cPPT: central polypurine tract; RRE:Rev response element; MND: MND LTR; SIN: Self inactivated; SV40USE: SV40upstream polyadenylation enhance element.

FIG. 5: A minimal BIV based vector system. The figure shows a minimalvector construct, a minimal packaging construct, and a minimal viralsurface protein expression construct.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, molecular biology,cell culture, virology, immunology and the like which are in the skillof one in the art. These techniques are fully disclosed in currentliterature and reference in made specifically to Sambrook, Fritsch andManiatis eds., “Molecular Cloning, A Laboratory Manual”, 2^(nd) Ed.,Cold Spring Harbor Laboratory Press (1989); Celis J. E. “Cell Biology, ALaboratory Handbook” Academic Press, Inc. (1994) and Bahnson et al., J.of Virol. Methods, 54:131-143 (1995).

All publications and patent applications cited in this specification areindicative of the level of skill of those skilled in the art to whichthis invention pertains and are hereby incorporated by reference intheir entirety.

As used in this specification and claims, the singular form “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, a virion particle includes a plurality of virionparticles. Polynucleotides or nucleic acids of the invention may be inthe form of RNA or in the form of DNA, which DNA includes cDNA, genomicDNA or synthetic DNA.

Bovine immunodeficiency virus (BIV) is classified in the retroviralsubfamily lentivirus (Gonda et al., Nature, 330:388-391 (1987)).Lentiviruses are exogenous, nononcogenic retroviruses and include equineinfectious anemia virus (EIAV), simian immunodeficiency viruses (SIVs),visna and progressive pneumonia viruses of sheep, felineimmunodeficiency virus (FIV) and human immunodeficiency viruses (HIV-1and HIV-2). BIV has immunologic cross-reactivity with HIV, SIV and EIAV.These viruses typically contain 8,000 to 10,000 nucleotides in theirgenome encompassing the gag, pol and env genes, as well as long terminalrepeat (LTR) sequences.

The “gag” gene is the 5′-most gene on retroviral genomes and encodesstructural proteins that form the virus particle. It is translated togive a precursor polyprotein that is subsequently cleaved to yield threeto five structural proteins.

The “pol” gene encodes enzymes needed for polyprotein cleavage, reversetranscription and proviral DNA integration into the chromosomes.

The “env” gene encodes the envelope proteins. As used in thisdisclosure, the env gene includes not only natural env gene sequencesbut also modifications to the env gene, including modifications thatalter target specificity of retroviruses or env genes that are used togenerate pseudotyped retrovirus. See PCT publication WO 92/14829,published Sep. 3, 1992, entitled “Viral Particles Having Altered HostRange.”

The retroviruses share features of the replication cycle, includingpackaging of viral RNA into virions, infection of target cells,production of a DNA copy of the RNA genome by reverse transcription ofthe RNA carried in the virion to form a DNA provirus, transport of theDNA to the cell nucleus, integration of the proviral DNA into the targetcell genome, transcription of mRNA from the viral DNA that is driven bya promoter within the 5′ LTR sequence, translation of the gag, pol andenv proteins, formation and maturation of viral particles and sheddingof single membrane enveloped virions from the cell. The LTR containscis-acting sequences important for reverse transcription, integration,transcription, and polyadenylation (Coffin J., J. Gen. Virology 42:1-26(1979)).

The genomes of the lentiviruses are complex. They share the abovementioned retroviral genes, gag, pol and env and in addition have anumber of nonstructural/regulatory genes including a “central region”(Braun et al., J. Virol. 61:4046-4054 (1987) and Chakrabarti et al.,Nature, 328:543-547 (1987)).

The complement of nonstructural/regulatory genes found in the genomes ofisolates from various species differ from one another, particularly inthe env sequences. The basic genomic organization of BIV is disclosed inGarvey et al., Virology, 175:391-409 (1990) and U.S. Pat. No. 5,380,830,the disclosure of which is incorporated herein by reference in itsentirety. Additionally disclosed are methods of obtaining BIV genomicDNA from BIV infected cells. The gag and pol genes are in differentframes and overlap. The pol and env genes are in the same reading frameand are separated by the “central region”. There are five open readingframes (ORFs) found in the central region. Three of these are similar instructure to the exons for vif, tat and rev of HIV and otherlentiviruses. The other two ORFs are located in a position in thecentral region analogous to vpr, vpx and vpu encoding ORFs of HIV-1and/or HIV-2. The nef ORF which is located post-env in the genomes ofother lentiviruses appears to be lacking in BIV (Garvey et al., 1990supra).

An exon is a segment of a gene. It codes for a specific part of theprotein. A gene is a nucleic acid segment that is involved in producinga polypeptide chain and includes regions preceding and following thecoding region segment of DNA.

The “tat” gene consists of two coding exons. The first is contained inthe central region and the second in the 3′ end of the env codingsequence but in a different reading frame from the env and rev exons.

The “rev” gene is also made up of two exons. The first is located nearthe 3′ end of the central region and overlaps the 5′end of env. Thesecond exon is found in the 3′ end of env but in a different readingframe. The “rev” gene product transports intron-containing viral mRNAs,including the full length RNA used for gag-pol translation and virionpackaging to the cytoplasm without splicing.

Sequences encoding BIV and plasmids containing retroviral genomessuitable for use in preparing the vector constructs of the invention maybe readily obtained given the disclosure provided herein or fromdepositories and databases such as the American Type Culture Collection(ATCC), for example, ATCC Accession No. 68092 and ATCC Accession No.68093 and GENBANK. Various BIV clones are disclosed in Garvey et al.,supra and in U.S. Pat. No. 5,380,830. Particular reference is made toBIV 127 and 106, having ATCC Accession Nos. 68092 and 68093,respectively.

It will be understood that for the nucleotide sequence of the BIVgenome, natural variations can exist between individual BIV viruses.These variations may result in deletions, substitutions, insertions,inversions or additions of one or more nucleotides as long as theclaimed function of the gene is not lost. The DNA sequences encodingsuch variants may be created by standard cloning methods or polymerasechain reaction (PCR) U.S. Pat. Nos. 4,683,195 and 4,683,202.

The present invention provides a nucleic acid segment from a BIV genomeobtainable from any strain or clone of BIV. In one embodiment, a BIVvector construct of the invention should include a sufficient number ofnucleotides corresponding to nucleotides of the BIV genome to expressone or more functional BIV genes.

A “BIV combination vector construct” refers to an assembly which iscapable of directing the expression of a nucleotide sequence andincludes a 5′ LTR region having a promoter sequence region which iscapable of initiating transcription of BIV; BIV gag and pol genesoperably linked to the promoter; a segment of the BIV env coding region;a BIV packaging sequence; and a transgene operably linked to a promoterand inserted into the segment of the env coding region. Preferably, thetransgene is operably linked to a second promoter which may be the sameor different from the promoter operably linked to the BIV gag and polgenes.

The BIV env gene found in BIV clone 127 is approximately 2711nucleotides long and starts at about nucleotide 5415. In a preferredembodiment, a transgene is inserted into the interior region of the envgene. For example the transgene may be inserted into the segment of theenv coding region after the tat and rev coding exon 1 segments andbefore the tat and rev coding exon 2 segments. Preferably, the transgeneis inserted after nucleotide 860 of the env coding segment whichcorresponds to BIV polynucleotide 6275. In another embodiment, a sectionof the env coding region is deleted. Preferably, the deleted section isfrom the interior region of the env gene and the transgene is insertedin the gap created by the deletion. The deleted section of the envcoding region may be as short as 20 nucleotides but may be as long as1500 nucleotides. Methods of deleting a section of the env gene are wellknown by those of ordinary skill in the art.

A “BIV vector construct” refers to an assembly which is capable ofdirecting the expression of a nucleotide sequence. The vector constructshould include a 5′ sequence (comprising a promoter region) which iscapable of initiating transcription of BIV; a DNA segment from a BIVgenome; a packaging sequence from a BIV or other lentivirus orretrovirus; and a transgene operably linked to a second promoter.Accordingly, in one aspect the present invention provides a BIV vectorconstruct comprising: a DNA segment from a BIV genome, a packagingsequence for packaging RNA into virions, a first promoter operablylinked to the DNA segment, and a transgene operably linked to a secondpromoter. In a preferred embodiment, the packaging sequence of the BIVvector construct is a BIV packaging sequence.

A portion of a BIV gag gene may be incorporated into the DNA segment.The BIV gag gene is approximately 1431 nucleotides. Preferably, theportion of the gag gene will include no more than about 200 nucleotides.The DNA segment may further comprise a BIV gene selected from the groupconsisting of vif, vpw, vpv, tat, rev, and env.

In another preferred embodiment, the transgene is operably linked to aCMV promoter, a PGK promoter, or a MND promoter. The BIV vectorconstruct may further comprise one or more of a rev response element(RRE), a interferon-beta scaffold attachment region (SAR), whichpreferredly is of human origin, and/or a central polypurine tract, whichpreferredly are located between the BIV 5′ segment and the internal(“second”) promoter. Thus, in one preferred embodiment, the transgeneoperably linked to a second promoter is located downstream of theputative BIV RRE.

In yet another preferred embodiment, the BIV vector construct of theinvention is a self-inactivating vector. In particular, a portion of theU3 region. of the 3′ LTR of the BIV vector construct may be deleted orreplaced by a heterologous sequence. Preferred is the replacement of aportion of the U3 region of the 3′ LTR by the SV40 late polyadenylationsignal enhancer element.

In a particularly preferred embodiment, the BIV vector construct is aself-inactivating vector and further includes a transgene which isoperably linked to a MND promoter and comprises a RRE and a centralpolypurine tract (cPPT) upstream of the MND promoter.

A “BIV packaging construct,” also sometimes referred to as a helperconstruct, refers to an assembly which is capable of directingexpression of a nucleotide sequence wherein the nucleotide sequenceincludes at least the gag gene or pol gene of BIV, a promoter operablylinked to the nucleotide sequence, and a polyadenylation sequencelocated downstream of the nucleotide sequence encoding the gag or polgenes. Preferably, the polyadenylation sequence is derived from Simianvirus 40 (SV40).

The BIV vector construct, BIV combination vector construct, and the BIVpackaging construct discussed above may include other BIV genes inaddition to the specific genes mentioned above. Other genes include pol,vif, vpw, vpv, tat, rev, and env genes. Particularly the BIV constructswould include nucleotides corresponding to a functional BIV rev gene.However, the BIV constructs may include a rev gene obtained from adifferent lentivirus and particularly an HIV rev gene. The BIVconstructs of the invention may also include a nuclear export element,preferably a rev response element (RRE), preferably downstream of thetransgene insert. The RRE may be from a lentivirus other than BIV.Preferably, the BIV constructs would also include a sufficient number ofnucleotides corresponding to a functional tat gene. The 3′LTR of the BIVgenome may also be included as described below.

In another embodiment, the present invention envisages a method ofproducing a packaging cell line which includes transforming, preferablytransfecting an established cell line with the BIV combination vectorconstruct or with a BIV packaging construct as disclosed above. For genetherapy applications, it is necessary to generate large volume ofsupernatants which can not be easily prepared by transient transfection.Therefore producer cell lines may be used to transfer genes of interestto a target cell. A producer cell line refers to a cell line which iscapable of producing recombinant BIV particles. The producer cell lineshould include an integrated BIV construct as disclosed above. A numberof BIV vectors may be contained within the recombinant BIV particle,including the BIV constructs of the present invention. Various methodsare known in the art to identify retroviral packaging cells capable ofproducing recombinant viral vector particles. These methods includeELISA and Western Blot.

Non-limiting examples of packaging cells lines include PA12, PA317,FLYA13, PE501, PG13, CRIP, RD114, GP7C-tTA-G10, PPA-6, and PT67 (Milleret al., Mol. Cell. Biol. 6:2895 (1986); Miller et al., Biotechniques7:980 (1989); Danos et al., Proc. Natl. Acad. Sci. USA 85:6460 (1988);Pear et al., Proc. Natl. Acad. Sci. USA 90:8392 (1993) and Rigg et al.,Virol. 218:290 (1996).

In an alternative preferred embodiment, the invention provides a minimalvector construct. The vector construct comprises a promoter linked to afirst BIV R region, a BIV U5 element linked to the first BIV R region, apackaging sequence, a transgene, and a BIV U3 element linked to a secondBIV R region, wherein the promoter initiates RNA transcription of thevector. Preferably, the packaging sequence is a BIV packaging sequence.Alternatively, it may be from another lentivirus or retrovirus. Inaddition, it is preferred that any start codons in the packagingsequence be eliminated by deletion or mutation. It is also preferredthat the major splice donor site be inactivated or eliminated. Thesechanges to the packaging sequence and the splice donor site areaccomplished by standard techniques.

In a particularly preferred aspect of the minimal vector, one or moresequences in the U3 element are mutated or deleted in order to diminishor eliminate U3-mediated transcription of any downstream genes. Thisprovides for a self-inactivating vector. In such a situation, thetransgene is operably linked to an internal promoter. In addition, it ispreferred that the U3 element further contain a sequence that enhancespolyadenylation. A preferred example is the SV40 late polyadenylationsignal upstream enhancer element (Sehet, et al., Mol. Cell Biol.,12:5386-5393 (1992)).

The minimal vector may contain additional elements. One such element isa cPPT. Preferably, the cPPT is a BIV cPPT. The vector may also furthercomprise a 3′. polypurine tract, which is located immediately upstream(i.e., 5′) of the 3′ U3 element.

In addition, the vector may comprise an RNA transport element. Suchtransport element may be a lentiviral RRE. Preferably, the lentiviralRRE is. a BIV RRE. Alternatively, the RNA transport element is aconstitutive transport element (CTE). Most preferably, the CTE is aMason-Pfizer Monkey Virus CTE. An alternatively preferred CTE is anAvian Leukemia Virus CTE.

In. an alternative preferred embodiment, the invention also provides aminimal packaging construct. The construct comprises a promoteroperatively linked. to a BIV gag/pol coding sequence and apolyadenylation signal at the 3′ end of the gag/pol coding sequence.Preferably, the packaging construct further contains a heterologousintron upstream (i.e., 5′) of the gag/pol coding sequence. In addition,the packaging construct may contain an RNA transport element. Thatelement may be a lentiviral RRE, preferably a BIV RRE, or it may be aCTE as described above. The packaging construct may also contain a Revcoding sequence.

A heterologous intron may be included in the constructs, andparticularly in the packaging constructs. Heterologous introns are knownand non-limiting examples include the human beta-globin gene intron.Introns used in the constructs of the invention may be obtained from theSV40 virus and the human insulin gene. Preferably, the intron will belocated upstream of the gag and pol nucleotide sequences.

In further embodiments, the constructs of the invention may include aninternal ribosome binding site (IRES), which allows translation of asecond gene, preferably a heterologous gene. The heterologous gene maybe a marker gene or a gene encoding a protein of interest as furtherdisclosed below.

A requirement for the expression of a BIV gene or a transgene is anadequate promoter operably linked to the coding nucleic acid sequence.The term “operably linked” refers to an arrangement of elements whereinthe components are configured so as to perform their usual or expectedfunction. The promoter or other control elements need not be contiguouswith the coding sequence. There may be intervening residues between apromoter or control elements and the coding region so long as thefunctional relationship is maintained.

With respect to the constructs as disclosed herein, the choice ofpromoter is well within the skill of one in the art and extends to anyeukaryotic, prokaryotic, or viral promoter capable of directing genetranscription in a target or host cell transformed with a constructaccording to the invention. The promoter may be a tissue specificpromoter, inducible promoter, synthetic promoter or hybrid promoter.More than one promoter may be placed in the constructs of the invention.Preferably, the BIV genes will be operably linked to one promoter andthe transgene insert will be operably linked to a second promoter.Examples of promoters useful in the constructs of the invention include,but are not limited to, phage lambda (PL) promoter; SV40 early promoter;a herpes simplex viral (HSV) promoter; a cytomegalovirus (CMV) promoter,such as the human CMV immediate early promoter; tetracycline-controlledtrans-activator-responsive promoter (tet) system; a long terminal repeat(LTR) promoter, such as a MoMLV LTR or an HIV LTR; the U3 regionpromoter of Moloney murine sarcoma virus; Granzyme A promoter;regulatory sequences of the metallothionin gene; CD34 promoter; CD8promoter; thymidine kinase (TK) promoter; B19 parovirus promoter; PGKpromoter; glucocorticoid promoters; heat shock protein (HSP) promoters,such as HSP65 and HSP70 promoters; immunglobulin promoters; MMTVpromoter; rous sarcoma virus (RSV) promoter; lac promoter; CaMV ³⁵Spromoters; and nopline synthetase promoter. Numerous promoters areavailable from commercial sources such as Stratagene (La Jolla, Calif.).

Preferred promoters include the promoter region of the LTRs, such as the5′ LTR promoter of HIV or BIV. Additionally preferred promoters includeCMV promoters and PGK promoters. Particularly preferred is the MNDpromoter. Various other control sequences may also be incorporated intothe constructs of the invention, for example enhancers and scaffoldattachment regions (SARs). Particularly preferred is the scaffoldattachment region derived from human interferon-beta. Particularenhancer sequences can readily be determined by one of ordinary skill inthe art.

A “packaging sequence” is defined as sequences necessary for packagingretroviral RNA into virions. The packaging sequences are generallylocated in the region between the 5′ major splice donor and the gag geneinitiation codon. While a BIV derived packaging sequence is preferred,the vectors according to the invention may include packaging sequencescorresponding to other retroviruses, particularly lentiviruses, and moreparticularly corresponding to HIV-1, HIV-2 or SIV. The packagingsequences may include as many as 1000 nucleotides or a few as 50nucleotides. The size of a packaging sequence region can be determinedby one of ordinary skill in the art.

As disclosed above, one or more constructs according to the inventionmay further include a polyadenylation signal (polyA) that is positioned3′ of the coding sequence. The polyA tail may be of any size which issufficient to promote stability in the cytoplasm. The polyA may bederived from a lentivirus such as BIV, HIV, and SIV. However, the polyAsequence may be derived from other viruses as well, such as the SV40polyA.

In addition to the promoter region, the long terminal repeats (LTR) ofretroviruses contain cis-acting elements that are important for reversetranscription, integration, transcription and polyadenylation, and oneor more of these elements may be incorporated into the constructs of theinvention. Preferably, the constructs comprise nucleotides correspondingto a sufficient number of nucleotides of a BIV LTR at the 5′ end toresult in a functional LTR. The constructs may also include a 3′LTRregion. The proviral LTR of BIV 127 is 589 polynucleotides. The LTR iscomprised of U3, R, and U5 elements. (U.S. Pat. No. 5,380,830).

The invention further encompasses two and three vector systems includingthe BIV vector construct, the BIV combination vector construct, and theBIV packaging construct. In one embodiment, the three vector system willcomprise the BIV vector construct and the BIV packaging vector constructas defined above and further a third construct. The third construct isan expression vector construct comprising a gene encoding a viralsurface protein from a different virus. The viral surface protein may beany other viral envelope protein, including vesicular stomatitis virusenvelope glycoprotein (VSV-G), MoMLV env protein, or envelopes derivedfrom Gibbon Ape Leukemia virus (GaLV), Rhabdoviride (Rabies, Mokola,Lyssa), Alphaviruses (Ross River virus, Sindbis), Paramyvovirus (Sendai)Filovirus (Ebola, Marburg), Retroviruses (MLV, 10A1, Xeno) Arenaviruses(LCMV), Parainfluenza virus. In a preferred embodiment the surfaceprotein is a VSV-G. (Burns et al., PNAS, 90:8033-8037 (1993) and Yee etal., PNAS 91:9564-9568 (1994)). See also U.S. Pat. No. 5,817,491, issuedOct. 6, 1998, the disclosure of which is incorporated herein byreference in its entirety.

A two-vector system may include the BIV combination vector constructaccording to the invention and the surface protein expression vector asdisclosed above. In a preferred embodiment the surface protein vectorconstruct is a vesicular stomatitis virus (VSV-G) envelope glycoproteinexpression vector.

The titer of VSV-G-pseudotyped BIV virus preparations can be increasedby centrifugation of the virus. An unconcentrated VSV-G-pseudotyped BIVvirus preparations may possess a titer of approximately 5×10⁵ infectiousunits (IU) per ml in the human kidney epithelial cell line 293T. Withvirus concentration, the titer can be raised significantly.

Generally, the titer of the vectors of the invention can be increasedby, first, collecting the virus particles via centrifugation of thevirus-containing medium, second, removing the supernatant, and, third,suspending the virus particles in a small volume of fresh medium. Forexample, concentrating the virus 10-fold by suspending the collectedvirus in a volume of liquid 10-fold smaller than the original volumebefore centrifugation, results in 3-10-fold increases in thetransduction efficiency. As the person skilled in the art will readilyappreciate, a further concentration of the virus will result in evenhigher increases in transduction efficiency.

The BIV based constructs according to the invention used alone or incombination may be used to transform virtually any cell line or hostcell. Eucaryotic host cells are preferred. Transformation may be bytransfection or transduction. Transfection is the transformation oftarget or host cells with isolated DNA genomes. Reference is made toKriegler, M., Gene Transfer and Expression: A Laboratory Manual, W.H.Freman & Company N.Y. (1990). Methods of transduction include directco-culture of cells with producer cells (Bregni et al., Blood80:1418-1422 (1992) or culturing with viral supernatant alone or with orappropriate growth factors (Xu et al., Exp. Hemat., 22:223-230 (1994).Reference is also made to commercially available kits, such as CaIPhoskit, (Clontech Inc. Palo Alto, Calif.) and Profection kit, (Promega,Madison Wis.) See Kotani et al., Human Gene Ther. 5:19-28 (1994) andForstell et al., J. Virol. Methods 60:171-178 (1996) for methods ofspinoculation.

Once a cell line is transfected or transduced with the constructs of theinvention, genes will be expressed and new virions will be made by thecell. As a result, the virions may be collected and used to infect atarget cell, thereby transferring the gene or genes of interest in thevector to the target cell.

Preferably, the cells are mammalian cells; most preferably, they areprimate cells, especially human cells. Examples include human embryonickidney cells (293T), EREp rabbit cells, Cf2Th (ATCC No. CRL 1430), CHO,SW480, CV-1, the human T cell line CEM-SS, Jurkat, the MDCK and D17 dogcell line, HT1090, LINA, WES and murine cell line NIH3T3. A cell line orcell culture denotes eukaryotic cells grown or maintained in vitro. Itis understood that descendants of a cell may not be completely identical(either morphologically, genotypically or phentotypically) to the parentcell.

In a particularly preferred embodiment of the invention, the minimalvector and packaging constructs are used to make packaging cells andproducer cells. The packaging cell comprises the packaging construct ofthe invention and a minimal viral surface protein expression construct.The latter construct comprises a promoter operatively linked to a viralenvelope coding sequence and a polyadenylation signal at the 3′ end ofthe coding sequence. The construct may further comprise a heterologousintron between the promoter and the coding sequence. The viral envelopeis any one of those previously described herein. Preferably, it is theVSV-G virus envelope.

The packaging cell is transfected. with the minimal vector construct tomake a producer cell. The producer cell comprises: (i) a BIV gag/polcoding sequence; (ii) a viral envelope coding sequence; and (iii) avector construct comprising a promoter linked to a first BIV R region, aBIV U5 element linked to the first BIV R region, a packaging sequence, atransgene, and a BIV U3 element linked to a second BIV R region. Theproducer cell is cultured, and it produces virions containing theminimal vector of the invention. This minimal vector is the RNA versionof the minimal vector construct and does not contain the promoter linkedto the first BIV R region. Preferably, the RNA vector further comprisesan internal promoter, as described. herein, operably linked to thetransgene. Most preferably, such vector is a SIN vector as describedherein. The virions are used to infect desired target cells, therebytransferring the transgene to the target cell.

A number of target cells, including cell lines and primary cells ofhuman and nonhuman origin, are successfully infected with the vectors ofthe invention. These include the dog osteosarcoma cell line D-17, therat smooth muscle cell line A-10, the mouse neuronal cell line MN9D, thehuman endothelial cell line HUVEC, rat primary endothelial cells, humanprimary lymphocytes and human primary hematopoetic stem cells. Thelatter two cell types are comprised mostly of non-dividing cells.Accordingly, the vectors of the present invention are particularlyuseful to infect non-dividing primary human cells. For example, thevectors of the invention can infect the HUVEC cell line aftergamma-irradiation to such an extent that proliferation of the cells wasabrogated. After such gamma-irradiation, the results are similar tothose before gamma-irradiation.

Preferred target or host cells are mammalian cells, preferably primatecells, and most preferably human cells. Preferred human cells includehematopoietic cells. Hematopoietic cells encompass hematopoietic stemcells, erythrocytes, neutrophils, monocytes, platelets, mast cells,eosinophils, basophils, B and T lymphocytes. Hematopoietic stem cellsand T-cells are especially preferred.

Prior to transfection or transduction, hematopoietic cells may beisolated and selected. Methods for isolating and selecting cells arewell known in the art (U.S. Pat. Nos. 5,061,620; 5,677,136 and5,750,397). For example sorted CD34⁺Thy-1⁺Lin⁻ cells from either adultbone marrow (ABM) or mobilized peripheral blood (MPB) are used.CD34⁺Thy-1⁺Lin⁻ are highly enriched in human hematopoietic stem cellsand can be isolated from ABM and MPB by flow cytometry (U.S. Pat. No.5,061,620).

The present invention also relates to a method of transferring a gene ofinterest to a mammalian cell comprising: transfecting a eukaryotic hostcell with the two or three vector system as disclosed above; culturingthe transfected host cell; collecting the virions produced; andadministering the collected virions to a mammalian cell to allowinfection of the mammalian cell and thereby transferring the gene ofinterest.

As disclosed above, methods of transfecting host cells are known andfurther reference is made to Graham and van der Eb, Virology52:456-467,1973.

Methods of culturing transfected and transduced cells are well known andreference is made to Freshney, R. I. “Culture of Animal Cells, A Manualof Basic Techniques”, Wiley-Liss Inc. (1994). Various culture media arecommercially available and non-limiting examples include DMEM, IMDM,X-vivo 15 and RPMI-1640. The formulations may be supplemented with avariety of different nutrients and growth factors. For culturing CD34⁺hematopoietic stem cells or progenitor cells, cytokines are preferablycombined in the media. These cytokines include but are not limited toIL-6, TPO, SCF, IL-3 and LIF. Methods of administering cells are wellknown, and these methods include administration. to human patients forgene therapy.

Methods of collecting virions produced by transfected cells aredescribed, for example, in Rigg et al., Virology 218:290-295. Thevirions, which include the BIV based constructs of the presentinvention, may be administered in vivo or in vitro to mammalian cells.Additionally, it is preferred that the virion includes a heterologoustherapeutic gene as disclosed herein. The virions produced according tothe invention by use of the BIV based constructs are recombinantparticles or virions. A “recombinant BIV particle or virion” refers to avirus particle that contains a BIV based vector RNA according to theinvention. In some instances, the BIV vector construct may be containedin a particle derived from viruses other than BIV, for example otherretroviruses and particularly other lentiviruses.

As used herein, the gene of interest generally referred to as atransgene is a heterologous gene, for example a marker gene. The choiceof a marker gene is within the skill of those in the art, andnon-limiting examples include, drug resistant markers, such as the(Neo^(r)) gene which encodes neomycin phosphotransferase; the HSV-tkgene, resulting in cells sensitive to agents such as acyclovir andgancyclovir; low-affinity nerve growth factor receptor (NGFR); enhancedgreen fluorescent protein (eGFP); enhanced yellow fluorescent protein;dihydrofolate reductase gene (DHFR); the bacterial hisD gene; murineCD24 (HSA); murine CD8a(lyt); bacterial genes which confer resistance topuromycin, phleomycin or beta-glactosidase (such as the lacZ gene); anda glutamine synthetase (GS) gene. In one preferred embodiment the markergene is eGFP. The marker gene is preferably under the control of aseparate promoter that will allow identification of target cellscontaining the marker gene.

A wide variety of nucleotide sequences generally referred to astransgenes may be carried by a BIV based construct of the presentinvention, in addition to or alternatively in the absence of a markergene. Preferably, the nucleotide sequences should be of sufficient sizeto allow production of viable virus particles. A non-exhaustive list ofthese transgenes (heterologous genes) includes sequences which encodeproteins, antigens, ribozymes, as well as antisense sequences.

The protein may be a therapeutic protein or a structural protein.Further the protein may be the entire protein or only the functionallyactive fragment thereof. The protein may include, for example, one thatregulates cell differentiation or a therapeutic gene capable ofcompensating for a deficiency in a patient that arises from a defectiveendogenous gene. The therapeutic gene may be one that antagonizesproduction or function of an infectious agent, antagonizes pathologicalprocesses, improves a host's genetic makeup or facilitates engraftment.

Specific examples of therapeutic genes or gene sequences are oneseffective in the treatment of adenosine deaminase deficiency (ADA);sickle cell anemia, recombinase deficiency, recombinase regulatory genedeficiency, HIV such as an antisense or transdominant REV gene, or genecarrying herpes simplex virus thymidine kinase (HSV-tk). The therapeuticgene may be a non-human gene, for example a yeast gene (Seo et al.,Proc. Natl. Acad. Sci. 95:9167 (1998)).

Nucleotide sequences for the transgenes may be obtained from variousdatabases such as GENBANK and from commercial sources such as AdvancedBiotechnologies (MD). Additionally cDNA sequences that encode for theheterologous sequence may be obtained from cells which express orcontain the sequence by methods well known in the art, utilizing forexample PCR.

Additionally the gene of interest may be selected from DNA sequencesencoding tumor necrosis factor genes, such as TNF-α; genes encodinginterferons such as interferon-α, interferon-β, and interferon-γ; genesencoding interleukins such as IL-1, IL-β, and interleukins 2 through 14,in particular IL-2, IL-4, IL-6 and IL-10; genes encoding GM-CSF orG-CSF; genes encoding adenosine deaminase, or ADA; genes which encodecellular growth factors, such as lymphokines, which are growth factorsfor lymphocytes; genes encoding soluble CD4; Factor VII; Factor IX;T-cell receptors; the LDL receptor, ApoE, ApoC, ApoAI and other genesinvolved in cholesterol transport and metabolism; the alpha-1antitrypsin gene, the ornithine transcarbamylase gene, the CFTR gene,the insulin gene, the NDI-1 gene, negative selective markers or“suicide” genes, such as viral thymidine kinase genes, such as theHerpes Simplex Virus thymidine kinase gene, the cytomegalovirus virusthymidine kinase gene, and the varicella-zoster virus thymidine kinasegene; Fc receptors for antigen-binding domains of antibodies, andantisense sequences which inhibit viral replication. Antisense sequencesare designed to bind RNA transcripts and thereby prevent cellularsynthesis of a particular protein or prevent use of that RNA sequence bythe cell.

For human patients, a therapeutic gene will generally be of human originalthough genes of closely related species that exhibit high homology andbiologically identical or equivalent function in humans may be used ifthe gene does not produce an adverse immune reaction in the recipient. Atherapeutic active amount of a nucleic acid sequence or a therapeuticgene is an amount effective at dosages and for a period of timenecessary to achieve the desired result. This amount may vary accordingto various factors including but not limited to sex, age, weight of asubject, and the like.

Methods known in the art may be used to assay the expression of atransgene in the target cell. These methods include flow cytometryselection, protein detection by western blot analysis, use of PCR (U.S.Pat. Nos. 4,683,195 and 4,683,202), nucleotide selection by northern orsouthern blots, and enzyme immunoassays, Sambrook J, Fritsch E F,Maniatis T. Molecular Cloning: A Laboratory Manual Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y., 1989.

EXAMPLES Example 1 Materials and Methods Example 1.1 Construction ofPlasmids

Vectors are generated differing in (i) the amount of gag and envsequences (BC vs. BC2 prefix); (ii) the internal promoter driving eGFPexpression: CMV (CG suffix) vs. PGK (phosphoglycerate kinase promoter(1); PG suffix) vs. MND (myeloid proliferative sarcoma virus promoter(Robbins, P. B., X. J. Yu, D. M. Skelton, K. A. Pepper, R. M. Wasserman,L. Zhu, and D. B. Kohn. 1997. J. Virol. 71:9466-9474); MG suffix); (iii)the placement of eGFP within the vector (i.e. upstream or downstream ofthe putative BIV RRE; BC2 vs. BC3 prefix); and (iv) additional segmentsinserted downstream of the putative BIV packaging signal (gag, ppt, sarsuffixes). In addition, two vectors contain a modified 3′ LTR in which alarge portion of U3 (including the TATA box) is replaced by a small SV40virus segment containing the late polyadenlyation signal upstreamenhancer element (USE; Schek, N., C. Cooke, and J. C. Alwine. 1992. MolCell Biol. 12:5386-5393; BC3 vs. BC4 prefix).

All restriction endonucleases are purchased from Roche MolecularBiochemicals (Indianapolis, Ind.). Plasmid pBIV, containing BIV proviralclone 127 (Garvey, K. J., M. S. Oberste, J. E. Elser, M. J. Braun, andM. A. Gonda. 1990. Virology. 175:391409), is obtained from the NationalInstitutes of Health.

Plasmid pCI is obtained from Promega (Madison, Wis.). Plasmid pCIGLcontains the vesicular stomatitis virus glycoprotein (VSV-G) cDNA(Burns, J. C., T. Friedmann, W. Driever, M. Burrascano, and J. K. Yee.1993. Proc Natl Acad Sci USA. 90:8033-8037, Yee, J. K., A. Miyanohara,P. Laporte, K. Bouic, J. C. Burns, and T. Friedmann. 1994. Proc NatlAcad Sci U S A. 91:9564-9568) in the pCI polylinker, downstream of thehuman cytomegalovirus (CMV) immediate-early promoter and chimeric intronand upstream of the simian virus 40 (SV40) late polyadenylation signal.

Plasmid pCrev, containing the HIV-1 rev cDNA under control of the CMVpromoter, has been described previously (Malim, M. H., J. Hauber, R.Fenrick, and B. R. Cullen. 1988. Nature. 335:181-183).

Plasmid pBH1 is generated by sequential insertion of two pBIV segmentsinto the pCI polylinker using standard techniques: a 5.5 kb SmaI-XbaIfragment containing the gag, pol, vif, vpw, and vpy genes, as well asthe first coding exons of the tat and rev genes; and a 1.3 kbDraIII-PvuII fragment containing the putative rev-response element (RRE)and the second coding exons of the tat and rev genes.

Plasmids pBH2 and pBH3 are constructed in the same way, but the chimericintron is deleted; moreover, the pBH25′ BIV fragment also contains the3′ 70 bp of the leader, including the major splice donor site.

Plasmids pBH1 and pBH3 are also modified by insertion of (1) an approx.500 bp fragment of HIV-1 containing the RRE, (2) an internal ribosomeentry site (IRES) from encephalomyocarditis virus (Jang, S. K., M. V.Davies, R. J. Kaufman, and E. Wimmer. 1989. J. Virol. 63:1651-1660), and(3) the puromycin N-acetyltransferase cDNA (Vara, J. A., A. Portela, J.Ortin, and A. Jimenez. 1986. Nuc Acids Res. 14:4617-4624).

Plasmid pBBB is generated by digesting plasmid pBIV with BfrI and BgIIIto remove most of the coding region and inserting into the gap a shortpolylinker created by annealing oligonucleotides BB5(5′-TTAAGATTTAAATACGCGTGCGGCCGCA-3′) and BB3(5′-GATCTGCGGCCGCACGCGTATTTAAATC-3′).

Packaging construct BH2 and an HIV-1 packaging construct (the HIVpackaging construct begins with the CMV promoter, followed by aheterologous intron, the HIV major splice donor, the entire HIV codingregion except for deletions in vpu, env and nef, then the HIV-1 RRE andfinally the SV40 polyadenylation site, see also Douglas, J., W.-Y. Lin,M. Panis, and G. Veres. Human Gene Therapy, “Efficient HIV-based vectortransduction of unstimulated human CD34+ cells in the SCID-hu Thy/Livmodel of human T cell lymphopoiesis.”) are each modified by insertion oftwo hemagglutinin (HA) oligonucleotides immediately upstream of the gagstop codon and deletion of most of the pol coding region as follows.First, a small fragment containing the gag stop codon of each packagingconstruct (290 bp ApaI-AccI fragment of BIV, 360 bp ApaI-BstXI fragmentof HIV-1) is ligated to ApaI/EcoRV-digested pBluescriptSK+ (Stratagene,La Jolla Calif.). Next, each subclone is subjected to reverse PCRamplification using primers containing the HA tags: HHAS5′-TATCCATACGATGTTCCAGATTATGCTTAAAGATAGGGGGGCAATTAAAG-3′ and HHAA5′-AGCATMTCTGGMCATCGTATGGATATTGTGACGAGGGGTCGCTG-3′ for HIV-1 and BHAS5′-TATCCATACGATGTTCCAGATTATGCTTAGACAAACAGCCTTTTTATAAAG-3′ and BHAA5′-AGCATMTCTGGMCATCGTATGGATAATCTAATATAAGAGGGGGTGC -3′ for BIV. Each PCRproduct is circularized, then the modified gag fragment is excised withApaI/SmaI and ligated to the parental packaging construct deletedbetween the ApaI site in gag and the 3′ end of pol (Asp718 for HIV-1,NsiI for BIV).

The CMV immediate-early enhancer/promoter is used to replace the BIVpromoter in the 5′ LTR in plasmids pBIV and pBBB as follows. First, theCMV region upstream of the TATA box is subjected to PCR amplificationwith primers CB5 (5′-CGGGATCCCGTAGTTATTMTAGTMTCMTTACGG-3′) and CMVBIV3(5′-AGATATGGTTTATATAGACCTCCCACCGTACA-3′) while the BIV region downstreamof the TATA box is subjected to PCR amplification with primers CMVBIV5(5′-GGGAGGTCTATATAAACCATATCTTCACTCTGT-3′) and Bgag3(5′-GCCGTTTCTGTACTCTCTGGT-3′). Second, the two amplified products aremixed and subjected to amplification using primers CB5 and Bgag3. Thefinal product is digested with XmaCI and ligated to plasmids pBIV andpBBB previously digested with NruI and XmaCI, generating plasmids pBIVCand pBBBC.

Plasmid pBIVC is then digested with SmaI and AfIIII to remove most ofthe coding region and blunt end-ligated to a DNA segment containingeither the CMV promoter or the mouse phosphoglycerate kinase (PGK)promoter (Adra, C. N., P. H. Boer, and M. W. McBurney. 1987. Gene.60:65-74) linked to the enhanced green fluorescent protein (eGFP) cDNA(Promega, Madison Wis.), generating plasmids pBCCG (also namedpBIVC-SACG) and pBCPG (also named pBIVC-SAPG), respectively.

Plasmid pBIVC is also digested with BfrI and BgIII to remove a smallersegment of the coding region, and then blunt end-ligated to the CMV-eGFPand PGK-eGFP cassettes, as well as a DNA segment containing eGFP linkedto the myeloid proliferative sarcoma virus (MND) promoter (Robbins, P.B., X. J. Yu, D. M. Skelton, K. A. Pepper, R. M. Wasserman, L. Zhu, andD. B. Kohn. 1997. J. Virol. 71:9466-9474), generating plasmids pBC2CG(also named pBIVC-BBCG), pBC2PG (also named pBIVC-BBPG), and pBC2MG,respectively.

Plasmid pBBBC is digested with MunI and HpaI to remove BIV sequencesbetween the putative RRE and the 3′ LTR, then ligated to the MND-eGFPand PGK-eGFP cassettes to generate plasmids pBC3MG and pBC3PG,respectively. The CMV-eGFP cassette is also inserted into the BstEIIsite of plasmid pBIV to generate pBCG.

Plasmid pBC3MG is digested with BfrI and BgIII to remove the shortpolylinker and then (1) ligated to a ˜500 bp BgIII fragment of the pBIVpol gene (containing the putative central polypurine tract) to produceplasmid pBC3MGppt; (2) ligated to a ˜1 kb BfrI fragment of pBIV(containing the 5′ end of the BIV gag gene) to produce plasmidpBC3MGgag; or (3) ligated to an ˜800 bp fragment containing the humaninterferon-β scaffold attachment region (SAR; Agarwal, M., T. W. Austin,F. Morel, J. Chen, E. Böhnlein, and 1. Plavec. 1998. 72:3720-3728) toproduce plasmid pBC3MGsar.

Plasmid pBC3MGgag is linearized with BfrI and ligated to the SAR andcPPT fragments to produce plasmids pBC3MGgagSAR and pBC3MGgagppt,respectively.

Plasmid pBC3MGppt is linearized with BfrI and ligated to the SARfragment to produce plasmids pBC3MGpptsar.

Plasmids pBC3 MP and pBC3 MPsar are generated from plasmids pBC3MG andpBC3MGsar, respectively, by replacement of the eGFP cDNA with thepuromycin N-acetyltransferase cDNA (Vara, J. A., A. Portela, J. Ortin,and A. Jimenez. 1986. Nuc Acids Res. 14:4617-4624).

Plasmids pBC4MG and pBC4MGppt, in which the 3′ LTR contains a largedeletion in the U3 region and an insertion of the SV40 latepolyadenylation signal upstream enhancer element (USE; Schek, N., C.Cooke, and J. C. Alwine. 1992. Mol Cell Biol. 12:5386-5393), aregenerated from plasmids pBC3MG and pBC3MGppt, respectively, as follows.First, the 5′ portion of the LTR and SV40 regions is subjected to PCRamplification with primers GFP5 (5′-GAGGACGGCAACATCCTGG -3′) and BSINSV3(5′-AGCAATAGCATCACAAATTTCACAAATAAACACATATGGGAAGTCCGGGG-3′) while the 3′portion is subjected to PCR amplification with primers BSINSV5(5′-GTGAAATTTGTGATGCTATTGCTTTATTTGTAATCTGTACTTCAGCTCGTGTAG-3′) and BIV3(5′-TCGCCGACATCACCGATGG-3′). Second, the two amplified products aremixed and subjected to amplification using. primers GFP5 and BIV3. Thefinal product is digested with SspBI and SphI and ligated to plasmidspBC3MG and pBC3MGppt previously digested with SspBI and SphI. Theresulting plasmids, pBC4MG. and pBC4MGppt, contain the 40 bp SV40 USE inplace of 332 bp of the U3 region.

For the relative assessment of the BIV gene transfer system, HIV-1 andMLV gene transfer systems are used. The construction and use of suchsystems for this purpose involve standard techniques known to thoseskilled in the art.

Example 1.2 Immortalized Cells

Cells are obtained from the following sources. 293T cells are obtainedfrom Gary Nolan (Stanford University, Palo Alto Calif.). CEMSS cells areobtained from the AIDS Reagent Program (Rockville Md.). A-10, and D-17cells are obtained from the American Tissue Type Collection (ATCC,Manassas Va.). MN9D cells (Choi, H. K., L. A. Won, P. J. Kontur, D. N.Hammond, A. P. Fox, B. H. Wainer, P. C. Hoffmann, and A. Heller. 1991.Brain Res. 552:67-76) are obtained from Rainer Ortmann (Novartis, Basel,Switzerland). Embryonal rabbit epithelial (EREp) cells (Oberste, M. S.,J. C. Williamson, J. D. Greenwood, K. Nagashima, T. D. Copeland, and M.A. Gonda. 1993. J. Virol. 67:6395-6405) is obtained from the NationalInstitutes of Health (Rockville Md.).

HUVEC (20) cells and primary rat aorta (smooth muscle) cells areobtained from Clonetics (San Diego Calif.). HUVEC cells are cultured inEBM basal media with the FGM bullet kit containing 0.1% human epidermalgrowth factor (hEGF), 2% fetal calf serum (FCS), 0.4% bovine brainextract w/heparin, 0.1% GA-1000 (gentamicin, amphotericin B), and 0.1%hydrocortisone.

Rat aorta cells are maintained in EBM basal media with the EGM-MV bulletkit containing 0.1% hEGF, 5% FCS, 0.4% bovine brain extract w/heparin,0.1% GA-1000, 0.1% hydrocortisone, and cultured in Primaria tissueculture flasks (Becton Dickinson Biosciences, San Jose Calif.).

CEMSS cells are cultured in RPMI-1640 medium supplemented with 10% FCS.All other cell lines are cultured in Dulbecco's modified Eagle medium(DMEM) supplemented with 10% FCS. 1.25×10⁵ HUVEC cells are suspended in5 ml of medium and irradiated with 8000 rad from a ¹³⁷Cs sourceirradiator (J. L. Shepherd, San Fernando Calif.), then transferred to a6-well dish and incubated for two days to allow for synchronization inthe G₂/M phase of the cell cycle.

Example 1.3 Virus Production

4-10×10⁶ 293T cells are seeded into 10 cm dishes overnight andtransfected the next day with 20-30 μg plasmid DNA by the calciumphosphate method (Clontech, Palo Alto Calif.). Typically, 20 μg of thevector, 10 μg of the packaging construct, and 3 μg of the VSV-G plasmidis used. In cases where the HIV-1 rev protein is required, 4 μg ofplasmid pCrev is added. 24-72 hours later, the cells or thevirus-containing medium is collected and analyzed in a variety of ways(see below). To assess the efficiency of transfection, a portion of thecells are analyzed for eGFP expression by flow cytometry, using aFACScan (Becton Dickinson Biosciences, San Jose Calif.). To measure theamount of virus shed into the medium, the medium is cleared of cellulardebris by low-speed centrifugation, then 10 μl is lysed and analyzed forRT activity using a commercial kit (Roche Molecular Biochemicals,Indianapolis, Ind.).

Example 1.4 Analysis of RNA Levels: Northern Assay

Transfected cells are lysed and cytoplasmic RNA is prepared using acommercial kit (Qiagen, Valencia Calif.). In addition, virus-containingmedium is collected, subjected to low-speed centrifugation to removecellular debris, and then subjected to high-speed centrifugation(50,000×g for 90 minutes at 4° C.) to collect the virus particles. Theviral pellet is lysed and the viral RNA is prepared using a commercialkit (Qiagen, Valencia Calif.). A fixed amount of cytoplasmic RNA (10 μg)or viral RNA (one third of the RNA preparation, not quantitated) issubjected to 1% agarose gel electrophoresis and transferred to a nylonfilter (Bio-Rad, Hercules Calif.). The filter is exposed to 40×10⁶ cpmof a DNA fragment random-primed with ³²P-dCTP using a commercial kit(Ambion, Austin Tex.), then washed and analyzed for bound probe with aphosphoimager (Molecular Dynamics, Sunnyvale Calif.). The probes includea BIV gag fragment, an HIV-1 gag fragment (both ˜1 kb in size), a ˜330bp BIV U3 fragment, and an ˜800 bp eGFP fragment.

Example 1.5 Analysis of Protein Levels: Western Assay

Transfected cells are lysed on ice for 1 hour in a buffer containing 1%NP-40, 150 mM NaCl, 10 mM Tris-Cl pH 7.4, 1 mM EDTA, and Pefablocprotease inhibitor (Roche Molecular Biochemicals, Indianapolis, Ind.).The lysate is subjected to centrifugation at 8,000×g for 20 minutes at4° C. to remove precipitated proteins and other debris. Alternatively,virus-containing medium is collected, subjected to brief centrifugationto remove cellular debris, and then subjected to high-speedcentrifugation (50,000×g 90 minutes at 4° C.) to collect the virusparticles. The viral pellet is lysed directly in SDS-PAGE sample buffer(Novex, San Diego Calif.). A fixed amount of cell or viral lysate issubjected to acrylamide gel electrophoresis and transferred to anitrocellulose filter. The filter is exposed to rabbit serum specificfor BIV Gag protein (obtained from the National Institutes of Health),then to horseradish peroxidase (HRP)-conjugated goat anti-rabbit Igantibody (Zymed, South San Francisco Calif.), then to the HRP substrateOPD (Sigma, St. Louis Mo.). Prestained molecular weight standards(Bio-Rad, Hercules Calif.) are used to determine the approximatemolecular weight of the BIV Gag bands. For the HIV western blot, thefilter is exposed to biotinylated anti-HIV Gag antibody (BeckmanCoulter, Fullteron Calif.) and then to steptavidin-conjugated HRP(Beckman Coulter, Fullteron Calif.) before the OPD reaction. For the HAwestern blot, the filter is exposed to a biotinylated anti-HA antibody(Roche Molecular Biochemicals, Indianapolis, Ind.), then to thesteptavidin-conjugated HRP before the OPD reaction. For the VSV-Gwestern blot, the filter is exposed to mouse anti-VSV-G monoclonalantibody (Sigma, St. Louis Mo.) and then to an HRP-conjugated goatanti-mouse Ig antibody (Zymed, South San Francisco Calif.) before theOPD reaction.

Example 1.6 Analysis of Transduction

To transduce cells, the virus-containing medium is subjected to briefcentrifugation to remove cellular debris, then 1 ml is added to freshcells in polypropylene tubes (5×10⁵ CEMSS cells) or in 6-well dishes(3-6×10⁵ adherent cells seeded per well the previous day). Protaminesulfate (Sigma, St. Louis Mo.) is added to the wells at a finalconcentration of 8 μg/ml and the tubes/dishes are subjected tocentrifugation (“spinoculation”) at 3000 rpm for 2-3 hours at 32-37° C.In later experiments, the tubes/wells are also supplemented with 10 mMHEPES buffer prior to spinoculation to prevent the pH of the medium fromrising while the cells are in the centrifuge. After spinoculation, thetubes and dishes are processed in different ways: supernatant isaspirated from the tubes, the CEMSS cells are suspended in fresh mediumand transferred to 6-well dishes. In contrast, the spinoculated dishesare placed back into the incubator for 30-60 minutes, then the medium isremoved and fresh medium is added to the wells. 2-3 dayspost-spinoculation, a portion of the cells are removed from the plateand analyzed for eGFP expression by flow cytometry, using a FACScan(Becton Dickinson Biosciences, San Jose Calif.). For virus preparationscontaining the vectors pBC3 MP and pBC3 MPsar, 293T cells are subjectedto spinoculation with serial dilutions of the virus-containing medium,and fresh medium supplemented with 5 μg/ml puromycin is added to thecells 24 hours post-spinoculation. 7-10 days later, colonies are countedby direct visualization.

Example 1.7 Infection of Primary T Cells

Human peripheral blood mononuclear cells (PBMC) are isolated from adultperipheral whole blood by Ficoll density gradient centrifugation, rinsedin phosphate-buffered saline (PBS), and suspended in RPMI-1640 mediumsupplemented with 10% FCS. A portion of the PBMC are activated by addingIL2 (Peprotech, Rocky Hill N.J.) to the medium at a final concentrationof 200 U/ml and culturing the cells for 3 days in 12-well dishes (3×10⁶cells per well) pre-coated as follows: the dishes are incubated with 1ug/ml goat anti-mouse Ig Fc (Pierce, Rockford Ill.) for 3 hours, rinsedwith PBS, incubated with a mixture of 1 ug/ml anti-CD3 (OKT3) and 10ng/ml anti-CD28 (BD Pharmingen, San Diego Calif.) monoclonal antibodiesfor 1 hour, and rinsed with medium. 5×10⁵ activated or unstimulated PBMCare spinoculated with viral supernatant in polypropylene tubes similarto CEMSS cells (see above); after spinoculation, the cells are rinsedand suspended in medium containing IL2 and cultured in 24-well dishespre-coated with the anti-CD3 and anti-CD28 mAbs. Four days and two weekslater, a portion of the cells are removed from the well and analyzed foreGFP expression by flow cytometry, using a FACScan (Becton DickinsonBiosciences, San Jose Calif.). T cells are identified by light scatterproperties and by expression of CD4 and CD8, using APC-conjugatedanti-CD4 and PerCP-conjugated anti-CD8 mAbs (Becton DickinsonBiosciences, San Jose Calif.).

Example 1.8 Infection of Hematopoietic Stem Cells (HSCs)

Human CD34⁺ cells are isolated from G-CSF-mobilized peripheral wholeblood from normal donors using Isolex 300SA (Baxter, Ill.). The cells(80-90% pure CD34⁺) are aliquoted (1×10⁷) and frozen in mediumconsisting of 45% Iscove's modified Eagle medium (IMDM), 45% FCS and 10%DMSO. Prior to transduction, the frozen cells are first thawed in buffercontaining 2% FCS, 1% HEPES, and 10 U/ml Heparin. After thawing, thecells (5×10⁵) are either spinoculated (see above) with viral supernatantin the absence of cytokines or cultured for 48 hours incytokine-containing medium (X-vivo15 medium (BioWhittaker, WalkersvilleMd.), thrombopoietin (tpo) mimetic (50 ng/ml; Novartis, Basel,Switzerland), flt3 ligand (100 ng/ml), and c-kit ligand (100 ng/ml; bothfrom Systemix, Palo Alto Calif.)) and then spinoculated with viralsupernatant. After infection, the cells are cultured incytokine-containing media. Three days or two weeks later, a portion ofthe cells is stained with APC-conjugated anti-CD34 antibody (BectonDickinson Biosciences, San Jose Calif.) and analyzed for eGFP on theCD34⁺ cells on a FACScan (Becton Dickinson Biosciences, San JoseCalif.).

Example 2 Transduction of Human Cells with BIV

The wild-type BIV genome is modified by insertion of the enhanced greenfluorescent protein (eGFP) marker gene, producing construct BCG (fordetails of plasmid construction see example 1). The eGFP gene isinserted into an interior position within the viral envelope gene so asnot to affect viral rev or tat expression or rev-response element (RRE)function. The human kidney carcinoma cell line 293T is cotransfectedwith construct BCG and a plasmid encoding the vesicular stomatitis virusglycoprotein (VSV-G); two days later, the virus-containing medium iscollected and exposed to fresh 293T cells. In addition, the medium isadded to the embryonal rabbit epithelial cell line EREp, which supportswild-type BIV replication (Oberste, M. S., J. C. Williamson, J. D.Greenwood, K. Nagashima, T. D. Copeland, and M. A. Gonda. 1993. J.Virol. 67:6395-6405.). Three days later, the exposed cells are assayedfor eGFP expression by flow cytometry. A subset of the 293T cells(approximately 5%) express eGFP, indicating that BIV can carry out allof the functions (including reverse transcription and integration)required for transduction of human cells. Similar transductionefficiencies are noted for the EREp cell line.

Example 3 BIV Packaging Constructs

Packaging constructs containing the viral genes are generated asdescribed in example 1 (FIG. 2) and assayed for gag mRNA expression andvirus production (Table 1) in the transient expression system. TABLE 1Virus production by the BIV constructs^(a) Con- struct expt#1 Expt#2expt#3 expt#4 expt#5 expt#6 expt#7 HIV 132 121 115 123 121 BIV 0 1 4BIVC 1 5 BH2 39 137 79 276 464 624 BH1 0 14 BH3 0 0^(a)Virus-containing media is collected from 293T cells transfected withthe indicated Construct and analyzed by RT assay. Seven transfectionsare performed. Values are given in pg of RT protein per ml media.

One construct, BIVC, is identical to wild-type BIV except for a precisereplacement of the 5′ LTR U3 region with the human cytomegalovirus (CMV)immediate-early promoter. The junction between the 2 segments is locatedat the identical TATA boxes to maximize the chances that the viral RNAwould contain the proper 5′ end for infection of target cells. The threeother BIV packaging constructs contain the CMV promoter linked to theBIV leader near of the gag gene, deleting the 5′ LTR and primer bindingsite and—in the case of constructs BH1 and BH3—the major splice donor.Construct BH1 contains a small chimeric intron inserted between the CMVand BIV segments. Downstream of the pol gene, construct BH2 contains allviral coding sequences except for a deletion (approximately 1 kb) of theinterior of env (not predicted to affect tat and rev expression or RREfunction), while constructs BH1 and BH3 contain BIV sequencesterminating approximately 250 bp downstream of the pol gene. Since BH1and BH3 lack the rev gene and the RRE, the HIV-1 RRE is inserteddownstream of the BIV sequences and HIV-1 rev protein is provided intrans when the constructs are characterized. In addition, these twoconstructs contain the puromycin N-acetyltransferase cDNA (Vara, J. A.,A. Portela, J. Ortin, and A. Jimenez. 1986 Nuc Acids Res. 14:4617-4624)coupled to an internal ribosome entry site (IRES) from theencephalomyocarditis virus (ECMV; Jang, S. K., M. V. Davies, R. J.Kaufman, and E. Wimmer. J. Virol. 63:1651-1660), for selecting celllines stably producing the packaging construct.

Northern analysis of transfected cell cytoplasmic RNA indicates thatconstruct BIVC produced higher steady-state levels of gag mRNA (816 cpm)than did wild-type BIV (249 cpm) or construct BCG (113 cpm), suggestingthat the CMV promoter is more active than the BIV LTR in 293T cells.Packaging constructs BH1 (70 cpm) and BH3 (39 cpm) express low levels ofgag mRNA, but BH2 (861 cpm) express high levels comparable to constructBIVC. For comparison, a CMV-driven HIV-1-derived packaging construct isobserved to express even higher levels of gag mRNA (1120 cpm).

Reverse transcriptase (RT) assay (Table 1) and western blot analysis ofvirus collected from the transfected cells indicate that BIVC producesmore virus particles than did wild-type BIV, BH1 and BH3, but much lessthan BH2 or the HIV-1 packaging construct. The low amount of BIVC virusmay have been due to toxicity in the transfected cells resulting fromBIVC's high-level expression of the wild-type BIV envelope protein, ascytopathic effects and small syncytia are observed in the culture.Western blot analysis indicates that both the BIVC and BH2 viruspreparations have undergone maturation, i.e. the Gag polyprotein isalmost entirely cleaved.

The RT assay indicates that the amount of virus produced by BH2 issimilar to that produced by the HIV-1 packaging construct. To verifythat the amounts of virus are similar, these two packaging constructsare modified by insertion of two consecutive hemagglutinin (HA) tagsimmediately upstream of the gag stop codon. The HA-tagged constructs,which are further deleted for most of the pol gene to block Gagpolyprotein cleavage, are introduced into 293T cells alongside theparental constructs. The modified virus is collected two days later andcompared to the parental virus by western blot assay using antibodiesspecific for Gag protein: both modified constructs produce amounts ofvirus similar to the parental constructs. The modified viruses are thennormalized by RT assay of the parental constructs (649 pg for HIV-1, 205pg for BH2) and analyzed for HA tag content by western blot assay. Theamounts of HA-tagged HIV-1 Gag polyprotein and HA-tagged BIV Gagpolyprotein are similar, corroborating the RT assay: the BH2 packagingconstruct produce levels of virus roughly similar to the HIV-1 packagingconstruct.

Western blot analysis is also performed on the BH2 and HIV-1 viruspreparations to assess the efficiency of VSV-G incorporation. Virus iscollected from 293T cells transfected with each packaging construct andthe VSV-G construct, then normalized by RT assay and subjected towestern blot analysis using a monoclonal antibody specific for VSV-G.The amount of VSV-G detected in the BH2 and HIV-1 samples is similar,indicating that BIV incorporates VSV-G as efficiently as HIV-1.

Example 4 BIV Vectors

BIV-derived vectors are generated, as described in detail in example 1,containing the eGFP marker gene and all viral elements required in cisfor transfer into target cells.

First, the two vectors (BCCG and BCPG) containing the full leader, butno gag sequences, and minimal env sequences (i.e. no RRE) are comparedto the analogous vectors (BC2CG and BC2PG) containing approximately 500bp of gag sequences and 1.2 kb of env sequences (including the putativeRRE). The latter vectors are generated because previous studies withother retroviruses have indicated that the 5′ end of the gag geneincreases the extent of vector RNA encapsidation in the virus particles,either by containing packaging elements or by stabilizing packagingelements further upstream (Bender, M. A., T. D. Palmer, R. E. Gelinas,and A. D. Miller. 1987. J. Virol. 61:1639-1646, Buchschacher, G. L., andA. T. Panganiban. 1992. J. Virol. 66:2731-2739; Parolin, C. P., T.Dorfman, G. Palu, H. Gottlinger, and J. Sodroski. 1994. J. Virol.68:3888-3895). In addition, studies with HIV-1 have indicated that thegag gene contains sequences which block RNA export from the nucleus, andthat the RRE removes this block in the presence of the rev protein(Malim, M. H., J. Hauber, S. Y. Le, J. V. Maizel, and B. R. Cullen.1989. Nature. 338:254-257; Schwartz, S., B. K. Felber, and G. N.Pavlakis. 1992. J. Virol. 66:150-159). The two vectors containing thePGK internal promoter are analyzed for transduction in 293T cells, usingthe BH2 packaging construct pseudotyped with VSV-G: BC2PG transduces 5%of the cells, while BCPG transduced none of the cells (FIG. 3, Exp.#1).All four vectors are then assessed for cytoplasmic RNA expression intransfected 293T cells and in collected BH2 virions by northern blotanalysis. Each of the vectors produces high levels of full-length vectorRNA in the transfected cell cytoplasm, but only the BC2CG and BC2PGfull-length RNAs are encapsidated efficiently by the BH2 virions.Therefore, the 5′ end of the BIV gag gene is likely to contain sequencesdirectly or indirectly required for viral RNA encapsidation.Interestingly, without those sequences on the BCPG and BCCG full-lengthRNAs, the internally-initiated mRNAs are encapsidated, suggesting thatother packaging elements reside in the 3′ portion of the BIV genome(Berkowitz, R. D., J. Fisher, and S. P. Goff. 1996. Current Topics inMicrobiology and Immunology. 214:177-218).

Next, the PGK promoter is compared to the MND promoter in two contexts,i.e. with the promoter-eGFP cassette upstream (BC2PG and BC2MG) ordownstream (BC3PG and BC3MG) of the putative BIV RRE. In 293T cells, thelatter context produces slightly higher transduction efficiencies thanthe former (14% vs. 6% for the PGK vectors and 35% vs. 29% for the MNDvectors), and in both contexts the MND promoter performs better than thePGK promoter (FIG. 3, Exp.#2). However, all vectors transducessubstantially fewer cells than did an HIV-1 virus containing ananalogous PGK-eGFP vector (75% transduction).

Virus containing the optimal vector, BC3MG, is then produced andanalyzed for its ability to be concentrated by centrifugation, for itsability to transduce a lymphoid cell line, and for the change in thefrequency of eGFP expression over two weeks post-infection. A portion ofthe virions are collected by centrifugation, suspended in a volume100-fold lower than the original volume, and then diluted either 10-foldor 100-fold. Although the 1× virus exhibits low transductionefficiencies, the T lymphoid cell line CEMSS is transduced moreefficiently (12%) than the 293T cells (5%). In addition, the 10× virustransduces a 10-fold higher percentage of 293T cells, and a 4.5-foldhigher percentage of CEM cells. Moreover, the percentage of eGFP⁺ cellsdecreases approximately 5-fold over two weeks in the 293T line, and to amuch lesser extent in the CEMSS line. Subsequent infections with new,unconcentrated virus preparations indicates that the fold-reduction inthe percentage of eGFP⁺ 293T cells over time correlates inversely withthe initial percentage of eGFP⁺ cells. For example, 293T cells which are73% eGFP⁺ 2 days post-infection are found to be 43% eGFP⁺ 2 weekspost-infection and 28% eGFP⁺ 4 weeks post-infection.

The higher transduction efficiencies exhibited by the BC3 vectorsrelative to the BC2 vectors may be due to a increased stability of thepackaging signal, due to its positioning farther away from non-viralsequences, i.e. the internal promoter (Kaye, J. F., J. H. Richardson,and A. M. L. Lever. 1995. J. Virol. 69:6588-6592). Additional viralsegments are therefore inserted immediately downstream of the gag regionin vector BC3MG, including an approximately 500 bp segment of the polgene, containing polypurine tracts that may function in an analogousmanner to the central polypurine tract (cPPT) region of HIV-1 (Charneau,P., M. Alizon, and F. Clavel. 1992. J. Virol. 66:2814-2820, Charneau,P., Mirambeau., R. G, P., S. Paulous, H. Buc, and F. Clavel. 1994. JMol. Biol. 241:651-662). In addition, the 3′ portion (approximately 1kb) of the gag gene is inserted: this vector (BC3MGgag) contains theentire gag gene except for approximately 200 bp in the capsid domain.The two new vectors are found to transduce slightly higher frequenciesof 293T cells (70% and 73%) than the parental vector BC3MG (55%) whenused with BH2 and VSV-G (FIG. 3, Exp.#3). For comparison, an HIV-1 viruscontaining an MND-eGFP vector transduces 100% of the cells.

The β-interferon scaffold attachment region (SAR), which has been shownto potentiate vector expression from integrated proviral DNA (Agarwal,M., T. W. Austin, F. Morel, J. Chen, E. Böhnlein, and 1. Plavec. 1998.Journal of Virology. 72:3720-3728), is also inserted immediatelydownstream of the packaging signal. This vector, BC3MGsar, transduces aslightly higher frequency of 293T cells than did the BC3MGppt vector(71% vs. 60%; FIG. 3, Exp.#4). In addition, vectors containing pairwisecombinations of the SAR segment, the 3′ gag segment, and the cPPTsegment are generated; however, none of these vectors exhibit highertransduction efficiencies than the vectors containing the individualsegments alone (FIG. 3, Exp.#4).

The BC3MG and BC3MGppt vectors are then compared to their SINcounterparts, BC4MG and BC4MGppt. These SIN vectors are deleted in theinterior 322 bp of the U3 region of the 3′ LTR, retaining 55 bp at the5′ end and 7 bp at the 3′ end; as a result, the TATA box and most of thepromoter elements are removed. Vectors deleted in this area are termed“self-inactivating”, or SIN, because the integrated vector in the targetcell possesses a 5′ LTR incapable of directing transcription (Miyoshi,H., U. Blomer, M. Takahashi, F. H. Gage, and 1. M. Verma. 1998. J.Virol. 72:8150-8157, Zufferey, R., T. Dull, R. J. Mandel, A. Bukovsky,D. Quiroz, L. Naldini, and D. Trono. 1998. J. Virol. 72:9873-9880). Thiseffect not only increases the safety of the vector, but in cases wheretranscription from the 5′ LTR interferes with transcription from thevector's internal promoter, the SIN deletion may also increase transgeneexpression in the transduced cell. It has also previously been reportedthat read-through transcription occurs from an integrated HIV-1 provirus(Dron, M., L. Hameau, L. Benboudjema, J. Guymarho, C. Cajean-Feroldi, C.Rizza PGodard, C. Jasmin, M. G. Tovey, and M. C. Lang. 1999. Arch Virol.144:19-28), suggesting that HIV-1 transcripts do not always terminate atthe 3′ LTR. Since this phenomenon might also occur in the BIV vectors,and might decrease the titer of the gene transfer system (Carswell, S.,and J. C. Alwine. 1989. Mol Cell Biol. 9:4248-4258), the SV40 latepolyadenylation signal upstream enhancer element (USE; Schek, N., C.Cooke, and J. C. Alwine. 1992. Mol Cell Biol. 12:5386-5393) is insertedinto the gap created by the SIN deletion. The two new vectors (BC4MG andBC4MGppt) are analyzed for transduction efficiency with packagingconstruct BH2 and VSV-G: BC4MG transduces 68% of the 293T cells, whileBC4MGppt transduces 90% (FIG. 3, Exp.#5). However, since the parental,non-SIN vectors exhibit similar transduction efficiencies (68% and 84%,respectively), the SIN deletion and SV40 USE insertion do notsubstantially increase the titer of the BIV gene transfer system.

To determine the titer of the BIV viruses with precision, the eGFP cDNAis removed from vectors BC3MG and BC3MGppt and replaced with thepuromycin N-acetyltransferase cDNA (Vara, J. A., A. Portela, J. Ortin,and A. Jimenez. 1986. Nuc Acids Res. 14:4617-4624), generating vectorsBC3 MP and BC3 MPppt. The same is done for the HIV-1 vector containingthe MND-eGFP cassette. The three viruses are prepared in 293T cells andserial dilutions are exposed to fresh 293T cells; after two days, thecells are treated with puromycin to kill the non-transduced cells. Aftera week in culture, the number of colonies growing in the dishes arecounted and used to calculate the titer of the original viruses. The HIVvirus titer is 1.2×10⁷ per ml, the BC3 MP virus titer is 3×10⁵ per mland the BC3 MPppt titer is 4.5×10⁵ per ml, nearly 30-fold lower than theHIV virus titer.

Example 5 Transduction of Other Cell Lines and Non-Dividing Cells

Concentrated BIV virus, prepared in 293T cells using packaging constructBH2, vector BC3MG, and VSV-G, is used to transduce a panel of celllines: D-17, a dog osteosarcoma line; A-10, a rat smooth muscle cellline; HUVEC, a human endothelial cell line; and MN9D, a mouse neuronalcell line. In each of these cell lines, the BIV virus transduced a largepercentage (63-88%) of the cells, even two weeks post-infection. Inaddition, primary rat endothelial cells are transduced by the BIV virus,albeit not as efficiently as the immortalized lines (22%).

To assess the ability of the BIV virus to transduce non-dividing cells,the concentrated BIV virus (10×) is assayed for transduction ofirradiated HUVEC cells and resting human peripheral blood lymphocytes(PBLs). The HUVEC line is irradiated two days before exposure to the BIVvirus, to synchronize the cells at the G₂/M phase of the cell cycle. Asexpected, a murine leukemia virus (MLV) virus preparation is observed toreadily transduce the untreated cells but not the irradiated cells. Incontrast, both the BIV and HIV-1 viruses transduce the untreated andirradiated cells with similar efficiencies, indicating that eachlentivirus is able to transduce the non-dividing cells efficiently.

In unstimulated human PBLs, the BIV virus exhibits transductionefficiencies similar to the HIV-1 virus when the cells are assayed fourdays after infection, although most of the BIV-transduced cellsexpresses very low levels of eGFP. Two weeks post-infection, however,these dim cells are nearly absent from the population. As a result, thepercentage of transduced cells is low: 1% for the 10× virus and 6% forthe 40× virus. However, the virus also exhibits low transductionefficiencies in pre-activated (i.e. proliferating) PBLs, as the 10×virus transduced only 5% of the cells. In contrast, 10× HIV-1 virustransduced 81% of the pre-activated cells and 44% of the unstimulatedcells, while 10× MLV virus transduced 43% of the pre-activated cells butonly 1% of the unstimulated cells.

Finally, the concentrated BIV virus is used to transduce unstimulatedmobilized peripheral CD34⁺ hematopoietic stem cells (HSCs), most ofwhich are quiescent (Knaan-Shanzer, F., D. Valerio, and V. W. vanBeusechem. 1996. Hum Gene Ther. 3:323-333 Uchida, N., D. He, A. Friera,M. Reitsma, D. Sasaki, B. Chen, and A. Tsukamoto. 1997. Blood.89:465-472). The 10× BIV preparation transduces 19% of the HSCs threedays post-infection, while the 40× BIV virus transduced 31% of thecells. Interestingly, almost all of the cells transduced with the 10×BIV virus express high levels of eGFP, and these cells do not disappearwhen the cells are analyzed 11 days later. The cells infected with the40× BIV virus contain two eGFP⁺ populations: one (18% of the cells)which expressed very low levels of eGFP and one (13% of the cells) whichexpressed higher levels of eGFP. Both populations exhibited 6-foldreductions in frequency over the next 11 days.

Example 6 BIV Based Gene Transfer System Example 6 Abbreviations

-   HIV: Human Immunodeficency Virus-   BIV: Bovine Immunodeficiency Virus-   VSV-G: Vesicular Stomatitis Virus Envelope Glycoprotein G-   MLV: Murine Leukemia Virus-   MV: Adeno-Associated Virus-   IU: Infectious Unit-   HSkMC: Human Primary Skeletal Muscle Cells

Example 6.2 Introduction

Various viral vectors have been explored as vehicles to delivertherapeutic genes for human gene therapy. Murine leukemia virus (MLV),adenovirus, and adeno associated virus (MV) based vectors have beenwidely used for such purposes. However, all these vector systems havetheir advantages as well as disadvantages. MLV based vectors have beenproven to be safe for human gene therapy, yet they suffer from aninability to transduce non-dividing cells, which are often thetherapeutic targets in vivo. Human adenovirus based vector are capableof efficiently transducing a variety of non-dividing target cells.However, a strong host immune reaction and transient gene expression haslimited the applications of the most commonly used avenovirus vectors.The low coding capacity of MV based vectors decreases the utility ofthese vectors.

Recently, lentiviruses based vector systems have been extensivelystudied for their potential use as a gene delivery system. Lentiviralvectors can efficiently transduce both dividing and non-dividing cells,and they stably integrate into host chromosomes, resulting in long-termgene expression. The vector system also offers wide tropism due to itsability to pseudotype with heterologous viral envelopes. In addition,lentiviral vectors potentially raise no concern over the issue of hostimmune reaction, owing to the fact the only the transgene is expressedin the target cells. In vitro, it has been shown that Humanimmunodeficiency virus (H IV) based VSV-G pseudotyped lentiviral vectorstransduce a variety of non-dividing cells, e.g. microphages, neurons,hepatic cells, photoreceptor cells, and hematopoetic stem cells. Invivo, lentiviral vectors have been shown to transduce rat neurons,resulting in long term transgene expression.

To circumvent the safety concerns related to HIV based lentiviralvectors, we have developed a Bovine Immunodeficiency Virus (B IV) basedlentiviral vector system. BIV is not a human pathogen and does not causeobvious disease in its natural host, cattle. We show here that 1) ourBIV based lentiviral vector has a titer of 1.2×10⁶ i.u./ml, which iscomparable with HIV based systems; 2) the BIV based vectors can beconcentrated more than 150 fold with a one ultracentrifugation step; and3) the BIV based vectors efficiently transduce both dividing andnon-dividing human primary skeletal muscle cells.

Example 6.3 Methods

Cells. Human primary skeletal muscle cells (hSkMC) were purchased fromClonetics (Clonetics, Walkersville, Md.) and maintained and culturedaccording to the manufacture's instructions. Human embryonic kidney 293Tcells were cultured in Dulbecco's Modified Eagle's medium (DMEM,BioWhittaker, Walkersville, Md.) containing 10% fetal bovine serum(complete DMEM medium) (FBS, Hyclone Labs, Logan, Utah).

BIV based gene transfer system. The BIV based gene transfer systemcontains three plasmid constructs: a BIV packaging construct to providethe helper function; a BIV lentiviral vector backbone encoding a markergene, such as eGFP, or a drug resistant gene, such as puromycinresistant gene; and an expression construct encoding the VSV-Gexpression gene. The BIV lentiviral vector backbone and the VSV-Gexpression construct are essentially the same as described above.However, the BIV packaging construct BH2 was modified. Specifically, a15 bp putative packaging sequence from the major splicing donor site(MSD) to BIV gag start codon was deleted, generating BH2Δψ. In order todelete the putative packaging sequence, the region between MSD to gagstart codon was subjected to PCR amplification with two primersPackageDel 5 (5′-CGACCCGGGCGGCCGCTTCG-3′) and PackageDel 3(5′-CTACTCACCTGTCCGGAGTC-3′). The amplified PCR product was digestedwith SmaI and ligated to BH2 plasmid previously digested with SmaIgiving rise to BH2Δψ.

Preparation of BIV lentiviral vector. To generate BIV lentiviral vector,85% confluent 293T cells in a 10 cm dish were transfected with 15 μgpackaging plasmid (BH2Δψ), 15 μg vector plasmid (BIVMNDeGFP orBIVMNDPuro), and 4.5 μg VSV-G expressing plasmid (pCMV.VSV-G) usingcasium phosphate based transfection system. Vector supernatant washarvested 48 hr post-transfection and filtered through a 0.45 μM filter.The vector was then aliquoted and stored at −80° C. until use. Toconcentrate BIV lentiviral vector, the vector supernatant harvested wassubjected to ultracentrifugation for 90 minutes at 100,000×g at 4° C.The pelleted vector was resuspended in a small volume of DMEM medium andaliquots were stored at −80° C. until use.

Transduction. Target cells were transduced with lentiviral vector forfour hours in the presence of 8 μg/ml protamine sulfate (Sigma, St.Luis, Mo.). The cells were then washed with cell culture medium twiceand continuously cultured for additional 48 hours. The transduced cellswere then analyzed for eGFP expression with FACScan.

Titration of BIV lentiviral vector. To titer BIV based lentiviralvector, 5×10⁴ 293T cells were plated in each well of a six-well plate onday one. On day two, a lentiviral vector (5 μl of nonconcentratedsupplemented with 2 ml of complete DMEM medium) encoding puromycinresistant gene was used to transduce the cells in the presence of 8μg/ml protamine sulfate. On Day three, the transduced cells weretrypsinized and 5% of the transduced cells from each well were plated ina 6 cm dish with 5 ml of complete DMEM medium in the presence of 5 μg/mlof Puromycin (Sigma, St. Luis, Mo.). Three days later, the medium waschanged with fresh cell culture medium with puromycin. Day sevenpost-transduction, the cell culture medium was aspirated and thepuromycin resistant clones were stained with coomassie blue and counted.

Reverse transcriptase assay. Taking advantage of the fact that BIV RTcross-reacts with HIV RT, we quantitated BIV RT with an HIV RT assay kitpurchased from Roche.

BrdU incorporation. To determine whether the cells were activelydividing when the cells were transduced, the cells were labeled withBrdU (BrdU labeling kit, purchased from Phamingen, San Diego, Calif.)according to the manufacture's instruction.

Example 6.4 Results

BIV based gene transfer system. The BIV based gene transfer systemconsists of a BIV packaging construct, BIV vector construct, and VSV-Gexpression construct (FIG. 4). To generate BIV lentiviral vector, thethree constructs were co-transfected into 293T cells, and the viralvector supernatant was harvested 48 hour post transfection as describedin Methods.

Titer of a BIV based lentiviral vector. In order to determine theapproximate titer of BIV based lentiviral vector, plasmid BH2Δψ (15 μg),BMNDpuro (a BIV lentiviral vector backbone with MND LTR promotingpuromycin resistant gene) (15 μg), and pCMV.VSV-G (4.5 μg) werecotransfected into 293T cells as described in Methods. As a control,BMNDpuro was replaced with BMNDeGFP (puromycin resistant coding gene inBMNDpuro was replaced with eGFP coding gene). The resulting BIVlentiviral vectors, encoding puromycin resistant gene or eGFP, were usedto transduce 293T cells. After puromycin selection, the puromycinresistant clones were counted. The vector achieved approximately 1.2×10⁶puromycin resistant clones per ml, suggesting that the BIV lentiviralvector has a titer of approximately 1.2×10⁶ i.u./ml. These clones wereBIVMNDpuro specific as no puromycin resistant clones were found in thecells transduced with BIVMNDeGFP.

Concentration of BIV based lentiviral vector. In order to obtain ahigher titer of BIV lentiviral vector, the vector supernatant wassubjected to untracentrifugation. The vector's RT activity before andafter concentration was measured and compared. As shown in Table 2, fromthree independent vector preparations, the RT activity increased by morethan 150 fold. TABLE 2 Concentration of BIV based lentiviral vectors.Three independent BIV lentiviral vector preparations were subjected toutracentrifugation. The Reverse Transcriptase (RT) activity was measuredbefore and after concentration. After one round of untracentrifugation,the RT activities were increased by more than 150 fold for all threeindependent preparations with two different BIV vectors. ReverseTranscriptase Activity Before Concen- Post Concen- Fold of BIV Vectortration (ng/ml) tration (ng/ml) Concentration BIVMNDLuc 22 4100 186BIVMNDeGFP 47 8130 172 BIVMNDeGFP 61 10200 167

Transduction of dividing human primary skeletal muscle cells by a BIVlentiviral vector. 1×10⁵ human primary skeletal muscle cells were platedin each well of a six-well plate. The next day, the cells were labeledwith BrdU or transduced with BMNDeGFP (120 ng RT equivalent for eachwell) for 3 hours in the presence of protamine sulfate as described inMethods. The transduced cells were analyzed for BrdU incorporation 16hours post-transduction or eGFP expression 48 hours post-transductionwith a flow cytometer. The hSkSMC were transduced efficiently with BIVbased lentiviral vector BMNDeGFP. More than 56% of the cells werepositive for eGFP expression with a relative mean eGFP intensity of morethan 2300, suggesting that BIV based lentiviral vector can efficientlytransduce human primary skeletal muscle cells. These cells were activelydividing at the time the cells were transduced as indicated by activeBrdU incorporation by the cells.

Transduction of non-dividing human primary skeletal muscle cells by aBIV lentiviral vector. To stop the cell cycling, hSkSMC were irradiatedwith gamma-irradiator at 3500 rads. 2×10⁵ irradiated cells were platedin each well of a six-well plate. The next day, the cells were labeledwith BrdU or transduced with BMNDeGFP (120 ng RT equivalent for eachwell) for 3 hours in the presence of protamine sulfate as described inMethods. The transduced cells were analyzed for BrdU incorporation 16hours post-transduction or eGFP expression 48 hours post-transductionwith a flow cytometer. The non-dividing hSkSMC were transducedefficiently with BIV based lentiviral vector BMNDeGFP. More than 59% ofthe cells were positive for eGFP expression with a relative mean eGFPintensity more than 1500, suggesting that BIV based lentiviral vectorcan efficiently transduce non-dividing human primary skeletal musclecells. These cells were confirmed to be non-dividing at the time thecells were transduced as indicated by a lack of BrdU incorporation.

Example 6.5 Discussion

Lentivirus based vectors have emerged as a promising gene transfertechnology for human gene therapy. Lentiviral vectors are able toefficiently transduce both dividing and non-dividing cells both in vitroand in vivo, resulting in long-term transgene expression. Sustainedlong-term therapeutic gene expression is required for certainapplications where life long gene expression is needed to achievetherapeutic effect. These include diseases such as neuronal diseases,metabolic disorders, and some ocular diseases. The majority of thesediseases have no effective therapies.

Although a variety of lentivirus based gene transfer systems have beendocumented, the most efficient system has been HIV based. Safety is ofparamount importance for viral vector based gene therapy. To circumventthe potential safety concerns related to HIV based lentiviral vectors,we have developed a BIV based gene transfer system. To our bestknowledge, BIV is not a human pathogen and does not cause any obviousdisease in its natural host.

BIV has not been intensively studied. For example, the packaging signalhad not been identified. As a consequence, in some prior constructs,both packaging and vector transcripts were packed into vector particles,resulting in lower titer BIV lentiviral vector. In this example, wedeleted a putative packaging sequence the region from the MSD to the gagstart codon. We found that this sequence is part of the BIV packagingsignal (data not shown). We have also found that the upstream sequenceof MSD and the first 200 bp of gag contain the additional elements forefficiently packaging BIV transcript.

We have determined the approximate titer of our current BIV lentiviralvector using a vector encoding puromycin resistant selection marker. TheBIV vector titer achieved approximate 1.2×10⁶ infectious units per ml,which is comparable with HIV based lentiviral vectors. We have alsodetermined that the VSV-G pseudotyped BIV lentiviral vector can beconcentrated to a higher titer with a simple ultracentrifugation step.

We have also confirmed that our BIV based lentiviral vector indeed isable to integrate into cellular chromosome, resulting long-termsustained gene expression as indicated by Southern blot analysis andcontinuous passage of BIV transduced human cells.

Skeletal muscle cells represent ideal targets for human gene therapyespecially for secretory therapeutic proteins as it is easy toadministrate an agent intramuscularly. In this study, we transduced bothdividing and non-dividing human primary skeletal muscle cells. We foundour nonconcentrated BIV based lentiviral vector efficiently transducedthese cells.

Example 6.6 Summary

Lentiviral based gene transfer systems represent a promising genedelivery technology due to their ability to efficiently transduce avariety of non-dividing target cells in vitro and in vivo. Incorporationinto the cellular chromosome results in long term transgene expression.In addition, lentiviral vector-mediated gene expression does not requirede novo synthesis of viral protein, reducing the potential eliminationof target cells by the host immune system. However, the lentivirussystems with the most promising results to date are those based on HIV.HIV is the causative agent of AIDS. Several animal lentivirus based genetransfer systems have been developed. Although these systems providealternatives to HIV based vectors, the titer and transduction efficiencyfrom animal lentiviral vectors are lower comparing to HIV based vectors.In this example, we described an improved BIV based lentiviral vectorsystem which achieved a titer 1×10⁶ i.u./ml (infectious units per ml)(nonconcentrated) and can be concentrated more than 150 fold. BIV basedlentiviral vectors efficiently transduce both dividing and non-dividinghuman skeletal muscle cells. Our data indicate that BIV based lentiviralvector may provide an excellent gene transfer system for human genetherapy.

Example 7 Generation of a Minimal BIV Based Lentiviral Vector System

A desired minimal BIV based lentiviral vector system consists of aminimal vector construct, a minimal packaging construct, and a viralsurface protein expression vector construct.

Example 7.1 Minimal Vector Construct

A minimal BIV based vector (designated BC4MG.min) contains a modifiedBIV 5′ LTR, mutated major splicing donor site, packaging sequence,minimal gag sequence, cPPT, a transgene operably linked to an internalpromoter, CTE (constitutive transport element from Mason-Pfizer monkeyvirus), 3′ PPT, and modified 3′ LTR. See FIG. 5. Specifically, theCMV-immediate-early promoter, ending in the TATA box is linked to theBIV 5′ LTR starting immediately after TATA box. BIV sequences terminateat 200 bp into gag coding region. All vectors contain a reporter gene orother transgene cDNA linked to a heterologous, internal promoter: CMV,PGK, or MND. Downstream of the transgene cDNA lies the BIV 3′ LTR and 80bp of adjacent env sequences that contains a 3′ PPT. A putative cPPT isinserted upstream of the internal promoter and one or multiple copies ofCTE is inserted downstream of the transgene. The 3′ LTR contains a largedeletion in the U3 region and an insertion of SV40 late polyadenylationsignal upstream enhancer element as described in Example 1.

The minimal BIV based vector is generated on the basis of BC4MGppt withthe following modifications: (i) further deletion of gag sequence to 200bp; (ii) mutation of gag start codon ATG; (iii) mutation of the majorsplicing donor site (MSD); and (iv) replacement of the putative BIVrev-response element (RRE) with a CTE.

To facilitate the plasmid manipulation, the BC4MGppt is digested withBspMI, and the resulting 4743 bp fragment containing the entire vectorsequence is cloned into pBluescript previously digested with HincII,giving rise to BS4MGppt.

To make a further deletion on gag sequence, BS4MGppt is digested withEcoNI and BqIII, the resulting 7287 bp fragment is self ligated to makeplasmid BS4MGppt.Δgag. The resulting vector contains only a 200 bp BIVgag sequence.

To mutate the major splicing donor site, plasmid BS4MGppt.Δgag from theupsteam of the CMV promoter region to the MSD region is PCR amplifiedwith primers MSD5 (5′-GCTCTAGAACTAGTGGATCCCCCGGCATCCCG-3′) and MSD3(5′-GGGGAAAACACGCAACTTCTCTCCTGTCCGGAG-3′). The amplified product isdigested with SpeI and SmaII and ligated into the 6373 bp fragmentresulting from BS4MGppt.Δgag previously digested with SpeI and SmaII.The resulting plasmid is named BS4MGppt.ΔMSD.

To make a mutation in the gag start codon, plasmid BS4MGpptΔMSD from theupstream of the CMV promoter region to the gag region is PCR amplifiedwith primers gag5 (5′-TCTAGAACTAGTGGATCCCCC-3′) and gag3(5′-CTCTTCAACCCGGGGAAAAC-3′). The amplified product is digested withSpeI and SmaII and ligated into the 6373 bp fragment resulting fromBS4MGppt.Δgag previously digested with SpeI and SmaII. The resultingplasmid is designated BS4MGppt.ΔgagATG.

To replace the putative BIV rev-response element (BIVRRE) with CTE, aCTE sequence is inserted into SspBI site of plasmid BS4MGppt.ΔgagATG andfollowed by complete removal of the BIVRRE. To insert the CTE, theMason-Pfizer Monkey Virus CTE (MPMV CTE) is PCR amplified with primersCTE5′ (5′-TGATCTGTACAAGTAAGCTAGCACCTCCCCTGTGAG-3′) and CTE3′(5′-CCTTTTGTACAGTCGACATGCATGACACATCCC-3′). The amplified product isdigested with SspBI and ligated into BS4MGppt.ΔgagATG digested withSspBI. The resulting plasmid is named BS4MGpptCTE.ΔgagATG. To removeBIVRRE, first, the upstream of the BIVRRE is subjected to PCRamplification with primers RRE1(5′-GTTGGCGCCCAACGTGGGGCTCGAGTAAGAGAG-3′) and RRE2(5′-TAAGTGACCTATTTCTTCAGTGGTGTGTGT-3′) while the downstream of theBIVRRE is subjected to PCR amplification with primers RRE3(5′-ATAGGTCACTTATATGGGAATGAAAGACCC-3′) and RRE4(5′-AACTGCTGAGGGCGGGACCGCATCTGG-3′). Second, the two amplified productsare mixed and subjected to PCR amplification with primers RRE1 and RRE4.The final product is digested with KasI and BbvCI and ligated to plasmidBS4MGpptCTE.ΔgagATG previously digested with KasI and BbvCI, generatinga minimal BIV based vector construct, BS4MG.min.

Example 7.2 Minimal Packaging Construct

A minimal packaging construct contains BIV gag and pol coding sequenceslinked to one or multiple copies of a CTE. See FIG. 5.

To generate the minimal packaging construct, first, CTE is PCR amplifiedwith two primers CTE1 (5′-CGGGGTACCACCTCCCCTGTGAGCTAG-3′) and CTE2(TGCTCTAGAGACACATCCCTCGGAGGC-3′). The amplified product is digested withKpnI and XbaI and ligated to a pCI plasmid previously digested with KpnIand XbaI, generating pCI.CTE. Second, BIV gag and pol coding sequence isPCR amplified with two primers GAG5(5′-CCGCTCGAGATGAAGAGAAGGGAGTTAGAA-3′) and POL3(5′-CCGCTCGAGTCACGAACTCCCATCTTGGAT-3′). The amplified product isdigested with XhoI and ligated to pCI.CTE previously digested with XhoI,generating a minimal BIV based packaging construct, BIVGPCTE.

Example 7.3 Expression Vector Construct

The minimal BIV based lentiviral vector system further contains a thirdconstruct, an expression vector construct comprising a gene encoding aviral envelope protein from a different virus. The viral surface proteinmay be any other viral envelope protein including vesicular stomatitisvirus envelope glycoprotein (VSV-G) and others. See FIG. 5.

The disclosures of all patents, publications (including published patentapplications), depository accession numbers, and database accessionnumbers are incorporated herein by reference to the same extent as ifeach patent, publication, depository accession number, and databaseaccession number were specifically and individually incorporated byreference.

It is understood, however, that the scope of the present invention isnot to be limited to the specific embodiments described above. Theinvention may be practiced other than as particularly described andstill be within the scope of the accompanying claims.

1-30. (canceled)
 31. A method of transferring a transgene to a mammaliancell comprising: transfecting a eukaryotic host cell with a vectorsystem of comprising: (a) a BIV vector construct comprising: a DNAsegment from a BIV genome, a packaging sequence to package RNA intovirions; a promoter operably linked to said segment; and a transgeneoperably linked to a second promoter, wherein at least one of vpw, vpyand tat is deleted from said BIV construct, (b) a BIV packagingconstruct comprising a BIV sequence comprising a gag gene, a pol gene orboth of BIV operably linked to a promoter, and a downstreampolyadenylation sequence; and (c) an expression construct comprising agene encoding a viral surface protein; culturing the transfected hostcell and collecting the virions produced; and administering thecollected virions to a mammalian cell to allow infection of themammalian cell and thereby transferring the transgene.
 32. The method ofclaim 31 wherein the mammalian cell is located in vitro.
 33. The methodof claim 31 wherein the mammalian cell is located in vivo. 34-78.(canceled)
 79. A method of transferring a transgene to a mammalian cellcomprising transfecting a eukaryotic host cell with a vector systemcomprising: a) a BIV vector construct comprising: a promoter linked to afirst BIV R region; a BIV U5 element linked to the first BIV R region; apackaging sequence; a transgene; and a BIV U3 element linked to a secondBIV R region, wherein the promoter initiates RNA transcription of thevector construct, and at least one of vpw, vpy and tat is deleted fromsaid construct b) a BIV packaging construct comprising: a BIV sequencecomprising a gag gene, a pol gene or both of BIV operably linked to apromoter, and a downstream polyadenylation sequence; and c) anexpression construct comprising a gene encoding a viral surface protein;culturing the transfected host cell and collecting the virions produced;and administering the collected virions to a mammalian cell to allowinfection of the mammalian cell and thereby transferring the transgene.80. (canceled)
 81. The method of claim 31, wherein said viral surfaceprotein is a VSV-G protein.
 82. The method of claim 31, wherein saidpackaging sequence is a BIV packaging sequence.
 83. The method of claim31 wherein said transgene is operably linked to a mammalian promoter.84. The method of claim 31, wherein said BIV vector construct furthercomprises a rev-response element (RRE).
 85. The method of claim 31,wherein said BIV vector construct further comprises a central polypurinetract (cPPT).
 86. The method of claim 79, wherein said transgene isoperably linked to an internal promoter.
 87. The method of claim 79,wherein one or more nucleotides in said U3 element are mutated ordeleted to diminish or eliminate U3-mediated transcription.
 88. Themethod of claim 79, wherein said packaging sequence is a BIV packagingsequence.
 89. The method of claim 79, wherein any start codons in saidpackaging sequence are eliminated by deletion or mutation.
 90. Themethod of claim 79, wherein said U3 element further comprises a sequencethat enhances polyadenylation.
 91. The method of claim 79, wherein saidvector construct further comprises a cPPT.
 92. The method of claim 91,wherein said cPPT is a BIV cPPT.
 93. The method of claim 79, whereinsaid vector construct further comprises a 3′ polypurine tract.
 94. Themethod of claim 79, wherein said vector construct further comprises anRNA transport element.
 95. The method of claim 94, wherein said RNAtransport element is a lentiviral rev response element (RRE).
 96. Themethod of claim 95, wherein said lentiviral RRE is a BIV RRE.
 97. Themethod of claim 94, wherein said RNA transport element is a constitutivetransport element (CTE).